Compounds and therapies for the prevention of vascular and non-vascular pathologies

ABSTRACT

The invention provides a method of treating a mammal having, or at risk of, an indication associated with a TGF-beta deficiency comprising administering one or more agents that is effective to elevate the level of TGF-beta. The invention also provides novel compounds that elevate TGF-beta levels, as well as pharmaceutical compositions comprising compounds that elevate TGF-beta levels, and methods for detecting diseases associated with endothelial cell activation.

PRIORITY OF INVENTION

This application is a divisional of U.S. patent application Ser. No.09/567,558, filed May 5, 2000, now U.S. Pat. No. 6,412,587 which is acontinuation of U.S. patent application Ser. No. 09/057,323, filed Apr.9, 1998 (the '323 application) now U.S. Pat. No. 6,117,911 which claimspriority under 35 U.S.C. §119(e) from U.S. Provisional PatentApplication No. 60/043,852, filed Apr. 11, 1997.

BACKGROUND OF THE INVENTION

TGF-beta dynamically regulates the differentiation of smooth musclecells, and has been postulated to maintain vessel wall structure.TGF-beta also appears to possess immunosuppressive properties whichprotect the vascular endothelium against local inflammation and damage.Moreover, TGF-beta may inhibit the proliferation and migration of smoothmuscle cells after vascular injury.

TGF-beta is synthesized as a latent peptide (FIG. 1). Latent TGF-betarefers to any of several complexes that include the 25 kD TGF-beta dimerin association with the latency associated peptide (LAP) or any ofseveral additional TGF-beta binding proteins (LTBPs). Latent TGF-betahas no biological activity, i.e., it does not bind to the TGF-betareceptors.

The 25 kD TGF-beta dimer is also found associated with matrix componentsor other plasma proteins (FIG. 1). TGF-beta that is associated withmatrix components or other plasma proteins is termed mature TGF-beta.This association also prevents the binding of TGF-beta to the TGF-betareceptors.

In addition to latent and mature forms of TGF-beta, which cannot bind tothe TGF-beta receptors and which possess no known biological activity,TGF-beta also exists in forms which are capable of binding to theTGF-beta receptors and which elicit biological effects (FIG. 1). Theseforms of TGF-beta are termed “active TGF-beta.” One example of a form ofactive TGF-beta is the 25 kD TGF-beta dimer which is free fromassociation with LAP/LTBPs, or matrix or plasma components. Theprocess(es) by which the latent form of TGF-beta is converted to theactive form is termed “activation.” The process(es) by which the matureform of TGF-beta is converted to the active form is termed “release.”

Decreased levels of TGF-beta have been implicated in the development ofatherosclerosis. Atherosclerosis is a disease of the major arteries,typified by changes in the vessel wall architecture. At lesion-pronesites where the endothelium becomes damaged or dysfunctional, smoothmuscle cells from the media of the vessel migrate into the intima. Atthese sites, leukocytes, and in particular, monocytes and macrophagesinvade the expanded intima. As the lesion develops, lipid from thecirculation is deposited into the intima (reviewed in Ross, Nature, 362801 (1993); Grainger et al. Biol. Rev. Camb. Philos. Soc., 70, 571(1995)).

Agents which elevate TGF-beta activity, such as tamoxifen (TMX)(Grainger et al., Biochem. J., 294, 109 (1993)) and aspirin (Grainger etal., Nat. Med., 1, 74 (1995)), can exhibit cardioprotective effects.However, the positive cardioprotective effects of these agents may becounterindicated by their potential side effects. TMX can cause livercarcinogenicity in rats, has been correlated with an increased risk ofendometrial cancer in women and may increase the risk of certain gutcancers. Aspirin may result in ulcerogenesis and increased bleeding.

Agents which elevate TGF-beta levels may also be useful to prevent ortreat diseases or conditions including cancer, Marfan's syndrome,Parkinson's disease, fibrosis, Alzheimer's disease, senile dementia,osteoporosis, diseases associated with inflammation, such as rheumatoidarthritis, multiple sclerosis and lupus erythematosus, and otherauto-immune disorders. Such agents may also be useful to promote woundhealing and to lower serum cholesterol levels.

Thus, there is a need for improved therapeutic methods and agents usefulto maintain or elevate TGF-beta levels in mammals.

SUMMARY OF THE INVENTION

The present invention provides a method to maintain or elevate TGF-betalevels in a mammal, such as a human, in need of such therapy. The methodcomprises administering an effective amount of an aspirinate as definedherein. The method can also be carried out by administering an amount ofa first therapeutic agent effective to elevate the level of latentTGF-beta and an amount of a second therapeutic agent effective toincrease the level of TGF-beta which is capable of binding to theTGF-beta receptors, wherein said amounts are effective to maintain orelevate the level of TGF-beta in said mammal.

The invention also provides a method of preventing or treating a mammal,such as a human, having, or at risk of, a vascular indication which isassociated with a TGF-beta deficiency. The method comprises theadministration of an amount of an aspirinate that elevates the level ofTGF-beta in said mammal so as to inhibit or reduce diminution in vessellumen diameter. Preferably, the levels of active TGF-beta are elevatedafter administration of the aspirinate.

Preferred agents useful in the practice of the invention are copperaspirinates. Preferably, the effective amount of aspirinate inhibitslipid accumulation, increases plaque stability, decreases lesionformation or development, promotes lesion regression, or any combinationthereof. Agents useful in the practice of the method include aspirinatesalts such as copper salts of aspirinates, including copper aspirinateitself (copper 2-acetylsalicylate or copper 2-acetoxybenzoate),salicylate salts such as copper salts of salicylates, including coppersalicylate (copper 2-hydroxybenzoate), or a compound of formula (I) (seebelow) including a pharmaceutically acceptable salt thereof, or acombination thereof.

An aspirinate useful in the present invention is a compound of formula(I):

wherein

R¹ is hydrogen, halo, nitro, cyano, hydroxy, CF₃, —NR_(c)R_(d),—C(═O)OR_(e), —C(═N)OR_(e) —OC(═O)OR_(e), (C₁-C₆)alkyl or (C₁-C₆)alkoxy;

R² is hydrogen or —XR_(a);

R³ is —C(═O)YR_(b), or —N(R_(f))C(═O)R_(g)—;

R⁴ is (═O)_(n); or R⁴ is (C₁-C₆)alkyl, (C₁-C₆)alkanoyl or(C₂-C₆)alkanoyloxy;

R⁵ is hydrogen, —C(═O)OR_(h) or —C(═O)SR_(h);

n is 0, 1 or 2;

X is oxygen, —N(R_(i))—, or sulfur;

Y is oxygen or sulfur;

R_(a) is (C₁-C₆)alkanoyl, (C₁-C₆)alkyl, or hydrogen;

R_(b) is hydrogen or (C₁-C₃)alkyl;

R_(c) and R_(d) are each independently hydrogen, (C₁-C₄)alkyl, phenyl,C(═O)OH, C(═O)O(C₁-C₄)alkyl CH₂C(═O)OH, CH₂C(═O)O(C₁-C₄)alkyl, or(C₁-C₄)alkoxy; or R_(c) and R_(d) together with the nitrogen to whichthey are attached are a 3, 4, 5, or 6 membered heterocyclic ring; and

R_(e)-R_(i) are independently hydrogen or (C₁-C₆)alkyl;

a pharmaceutically acceptable salt thereof; or a combination thereof;

provided that R² and R³ are on adjacent positions of the ring to whichthey are attached, or are on the 2- and 5-positions of the ring; andfurther provided that when R² is hydrogen; R³ is on the 2-or 5-positionof the ring to which it is attached and R⁴ is (C₁-C₄)alkanoyloxy.Preferably, the compound of formula (I) is not3-acetoxy-2-carboxythiophene.

Also provided is a method of preventing or treating a mammal having, orat risk of, a vascular indication by administering there to an amount ofa first therapeutic agent and an amount of a second therapeutic agentwhich together are effective to elevate the level of TGF-beta,preferably the level of active TGF-beta, in said mammal. Preferably, theadministration inhibits or reduces diminution in vessel lumen diameter.The inhibition or reduction in diminution in vessel lumen diameterpreferentially occurs at a site in a vessel where the vascularindication is, or is likely to be, manifested. The invention thusprovides for combination therapy, e.g., the administration of one agentthat can elevate the level of latent TGF-beta, and another agent thatcan elevate the level of TGF-beta which is available to bind to, or iscapable of binding to, the TGF-beta receptor. This combination therapycan yield a significantly greater cardiovascular efficacy than would beexpected from the administration of either agent singly. The therapeuticagents can act in a synergistic, rather than in an additive, manner toelevate TGF-beta levels. The therapeutic agents can be administeredsimultaneously in a single dosage form simultaneously in individualdoses, or sequentially.

A first therapeutic agent useful in this embodiment of the inventionincludes an aspirinate, e.g., a compound of formula (I). Anotherpreferred first therapeutic agent comprises a compound of formula VI(see below). A preferred second therapeutic agent useful in thisembodiment of the invention comprises at least one omega-3 fatty acid,which can be provided, e.g., by dosages of fish oil. Another preferredsecond therapeutic agent is selected from at least one compound offormula VI. Thus, a compound of formula VI may both elevate latentlevels of TGF-beta and elevate the levels of TGF-beta which can bind tothe TGF-beta receptors. Preferably, the combination of the therapeuticagents inhibits lipid accumulation, increases plaque stability,decreases lesion formation or development, promotes lesion regression,or any combination thereof.

A compound useful in the present invention is a compound of formula(VI):

wherein

R⁶ is (C₁-C₆)alkyl, or aryl, optionally substituted by 1, 2, or 3 V;

R⁷ is phenyl, optionally substituted by 1, 2, or 3 V; or R⁷ is(C₁-C₁₂)alkyl, halo(C₁-C₁₂)alkyl, (C₁-C₆)cycloalkyl,(C₁-C₆)alkylcyclo(C₁-C₆)alkyl, (C₁-C₆)cycloalkenyl, or(C₁-C₆)alkyl(C₁-C₆)cycloalkenyl;

R⁸ is hydrogen or phenyl, optionally substituted at the 2-position withR_(j), and optionally substituted by 1, 2, or 3 V;

R⁹ is hydrogen, nitro, halo, aryl, heteroaryl, aryl(C₁-C₃)alkyl,heteroaryl(C₁-C₃)alkyl, halo(C₁-C₁₂)alkyl, cyano(C₁-C₁₂)alkyl,(C₁-C₄)alkoxycarbonyl(C₁-C₆)alkyl, (C₁-C₁₂)alkyl, (C₁-C₆)cycloalkyl,(C₁-C₆)alkylcyclo(C₁-C₆)alkyl, (C₁-C₆)cycloalkenyl, or(C₁-C₆)alkyl(C₁-C₆)cycloalkenyl, wherein any aryl or heteroaryl mayoptionally be substituted by 1, 2, or 3, V; or

R⁹ and R_(j) together are —CH₂CH₂—, —S—, —O— —N(H)—, —N[(C₁-C₆)alkyl]—,—OCH₂—, —OC[(C₁-C₆)alkyl]₂—, or —CH═CH—;

— is a single bond or is —C(B)(D)—, wherein B and D are eachindependently hydrogen, (C₁-C₆)alkyl, or halo;

V is OPO₃H₂, (C₁-C₆)alkyl, (C₁-C₆)alkoxy, mercapto, (C₁-C₄)alkylthio,halo, trifluoromethyl, pentafluoroethyl, nitro, N(R_(n))(R_(o)), cyano,trifluoromethoxy, pentafluoroethoxy, benzoyl, hydroxy,—(CH₂)₀₋₄C(═O)(C₁-C₆)alkyl, —UC(═O)(C₁-C₆)alkyl, benzyl,—OSO₂(CH₂)₀₋₄CH₃, —U(CH₂)₁₋₄COOR_(p), —(CH₂)₀₋₄COOR_(p),—U(CH₂)₂₋₄OR_(p), —(CH₂)₀₋₄OR_(p), —U(CH₂)₁₋₄C(═O)R_(k),—(CH₂)₀₋₄C(═O)R_(k), —U(CH₂)₁₋₄R_(k), —(CH₂)₀₋₄R_(k), or—U(CH₂)₂₋₄OC(═O)R_(p); wherein U is O, N(R_(m)), or S;

Z is —(CH₂)₁₋₃—, O, —OCH₂—, —CH₂O—, —C(═O)O—, —N(R_(q))—, C═O, or acovalent bond;

R_(k) is amino, optionally substituted with one or two (C₁-C₆)alkyl; oran N-heterocyclic ring optionally containing 1 or 2 additional N(R1), S,or nonperoxide O, wherein R₁ is H(C₁-C₆)alkyl, phenyl, or benzyl;

R_(n) and R_(o) are independently hydrogen, (C₁-C₆)alkyl, phenyl,benzyl, or (C₁-C₆)alkanoyl; or R_(n) and R_(o) together with thenitrogen to which they are attached are a 3, 4, 5, or 6 memberedheterocyclic ring;

R_(p) is H or (C₁-C₆)alkyl; and

R_(m) and R_(q) are independently hydrogen, (C₁-C₆)alkyl, phenyl,benzyl, or (C₁-C₆)alkanoyl;

the compound is MER25;

or a pharmaceutically acceptable salt thereof.

As described hereinbelow, the combination of aspirin plus an agent suchas fish oil that increases the level of TGF-beta which is capable ofbinding to the TGF-beta receptors, results in a greater reduction inlesion formation in apoE knockout mice relative to aspirin or fish oiltherapy alone. Surprisingly, the combination of aspirin and fish oil,which comprises a plurality of omega-3 fatty acids, exerts a markedlysynergistic, rather than an additive, effect. Thus, a combination of anagent that elevates the level of latent TGF-beta, e.g., low doses ofaspirin or an aspirinate, with an agent that increases the level ofTGF-beta which can bind to its receptor, e.g., at least one omega-3fatty acid, can be very effective in preventing or treating vasculardisease. As used herein, “at least one” omega-3 fatty acid reflects thefact that one of skill in the art would recognize that natural sourcesof omega-3 fatty acids contain a plurality, about 1 to 30, preferablyabout 1 to 25, and more preferably about 2 to 20, of omega-3 fattyacids.

Another embodiment of the invention is a method for preventingatherosclerosis in a mammal at risk therefor, or treatingatherosclerosis in a mammal, by administering to the mammal an amount ofa first therapeutic agent and an amount of a second therapeutic agenteffective to maintain or elevate the level of TGF-beta. The firsttherapeutic agent preferably increases the level of latent TGF-beta,e.g., is aspirin or an aspirinate, or a combination thereof, and thesecond therapeutic agent increases the level of TGF-beta which iscapable of binding to the TGF-beta receptors. Thus, the agents of theinvention are administered in a combined amount that prevents orinhibits diminution in vessel lumen diameter at, or near, a site orpotential site of atherosclerotic lesion formation or development. Apreferred first therapeutic agent comprises aspirin or an aspirinate. Apreferred second therapeutic agent comprises at least one omega-3 fattyacid.

The invention also provides a method to inhibit diminution in mammalianvessel lumen diameter. The method comprises administering to a mammal inneed of said therapy, an amount of a first therapeutic agent and anamount of a second therapeutic agent effective to maintain or elevatethe level of TGF-beta, so as to inhibit or reduce vessel lumendiminution. The inhibition or reduction in diminution in vessel lumendiameter preferentially occurs at a site in a vessel where thediminution is or is likely to be manifested. The first therapeutic agentincreases the level of latent TGF-beta, with the proviso that the firsttherapeutic agent is not aspirin. The first therapeutic agent ispreferably an aspirinate. The second therapeutic agent increases thelevel of TGF-beta which is capable of binding to the TGF-beta receptors.

Also provided is a combination therapy to maintain or elevate TGF-betalevels in a mammal in need of such treatment. The method comprises theadministration of an amount of a first therapeutic agent and a secondtherapeutic agent, wherein said amount is effective to maintain orelevate the level of TGF-beta. The first therapeutic agent increases thelevel of latent TGF-beta, while the second therapeutic agent increasesthe level of TGF-beta which is capable of binding to the TGF-betareceptors. A preferred first therapeutic agent comprises aspirin or anaspirinate, while a preferred second therapeutic agent comprises atleast one omega-3 fatty acid.

The invention also provides a method to maintain or elevate TGF-betalevels in a mammal in need of such treatment. The method comprises theadministration of an amount of an aspirinate effective to maintain orelevate the level of TGF-beta, preferably active TGF-beta, in saidmammal.

The invention also provides a method of preventing or treating a mammalhaving, or preventing in a mammal at risk of, a condition which isassociated with a TGF-beta deficiency. Also provided is a method tomaintain TGF-beta levels in a mammal. The methods comprise theadministration of one or more agents in an amount effective to elevateor maintain the level of TGF-beta in said mammal. The effective amountof the agent or agents may increase the level of latent TGF-beta or thelevel of TGF-beta which is capable of binding to the TGF-beta receptors.Agents useful to increase the level of latent TGF-beta include, but arenot limited to, idoxifene, toremifene, raloxifene, droloxifene, ethynylestradiol, diethylstibestrol, 1,25 dihydroxy-vitamin D3, retinoic acidand ligand pharmaceutical analogs thereof (Mukherjee et al. Nature,1997, 386: 407-410), dexamethasone, progesterone, thyroid hormoneanalogues (e.g. sodium liothyronine and sodium levothyroxine),hexamethylene bisacetamide, 4-hydroxyquinazoline, coumarin andbenzocaine.

Agents useful to increase the level of TGF-beta which is capable ofbinding to the TGF-beta receptors include agents that cause the releaseof TGF-beta from matrix components or plasma proteins, e.g., agents suchas heparin sugar analogs and betaglycan proteoglycan chains, or causethe release of TGF-beta from lipoproprotein complexes, e.g., agents suchas vitamin E, simvastatin, VLDL-lowering agents, Apo-AII-loweringagents, and ApoAI-stimulating agents. Other agents useful to increasethe level of TGF-beta which is capable of binding to the TGF-betareceptors include agents that cause an increase in the conversion of thelatent form of TGF-beta to the active form of TGF-beta, e.g.,hydrocortisone, dexamethasone, compounds of formula VI, vitamin D3,retinoic acid, simvastatin and thrombospondin.

Also provided is a kit comprising packing material enclosing, separatelypackaged, at least one device adapted for the delivery of a unit dosageform of a therapeutic agent and at least one unit dosage form comprisingan amount of at least one of the therapeutic agents of the inventioneffective to accomplish at least one of the therapeutic resultsdescribed herein when administered locally or systemically, as well asinstruction means for its use, in accord with the present methods. Asused herein, a “device adapted for delivery” of a therapeutic agentincludes, but is not limited to, a catheter, a stent, a stet, a shunt, asynthetic graft, and the like.

Also provided is a kit comprising packing material enclosing, separatelypackaged, at least one device adapted for the delivery of a therapeuticagent to a site in the lumen of a mammalian vessel and at least one unitdosage form of a first therapeutic agent and one unit dosage form of asecond therapeutic agent effective to accomplish at least one of thetherapeutic results described herein when administered locally orsystemically, as well as instruction means for its use, in accord withthe present methods.

Further provided is a pharmaceutical composition comprising a) at leastone aspirinate, and b) at least one omega-3 fatty acid, whereincomponents (a) and (b) are present in a combined amount effective tomaintain or increase TGF-beta levels, preferably at or near a site, orpotential site, of atherosclerotic lesion formation or development.

The invention also provides a pharmaceutical composition comprising (a)an amount of a first agent effective to elevate the level of latentTGF-beta; and (b) an amount of a second agent effective to increase thelevel of TGF-beta which is capable of binding to the TGF-beta receptors.

The invention also provides a pharmaceutical composition comprising a)an aspirinate, such as copper 2-acetylsalicylate or a compound offormula (I), and b) a compound of formula (VI), wherein components (a)and (b) are present in a combined amount effective to maintain orincrease TGF-beta levels, preferably at or near a site, or potentialsite, of atherosclerotic lesion formation or development.

Also provided are novel compounds of formula (I), (II), (III), (IV),(V), (VI), (VII), or (VIII) or pharmaceutically acceptable saltsthereof, and pharmaceutical compositions comprising a novel compound offormula (I), (II), (III), (IV), (V), (VI), (VII), or (VIII) as describedherein or a pharmaceutically acceptable salt thereof, which are usefulalone, or in combination, to elevate the level of TGF-beta in a mammal.

The invention also provides a therapeutic method. The method comprisesidentifying a patient exhibiting a decreased level of active TGF-betaand afflicted with a pathology associated with said decreased level. Thepatient so identified can be treated with an agent that elevates thelevels of active TGF-beta so as to alleviate at least one of thesymptoms of said pathology.

The invention also provides a method comprising determining endothelialcell activation in a mammal by detecting immunoglobulins thatspecifically bind to a TGF-β Type II receptor or a portion thereof.

The invention also provides a method comprising diagnosing or monitoringa disease characterized by endothelial cell activation (e.g.atherosclerosis) in a mammal by detecting immunoglobulins thatspecifically bind to a TGF-β Type II receptor or a portion thereof.

The invention also provides a method comprising detecting mammaliancells having TGF-β Type II receptors, by combining the cells with acapture moiety that binds TGF-β type II receptors or a portion thereof,forming a capture complex; and detecting or determining the amount ofthe capture complex.

The invention also provides a kit comprising packaging materialcontaining: a) a capture moiety comprising the extracellular domain ofthe TGF-β Type II receptor; and b) a detection moiety capable of bindingto an immunoglobulin. The invention also provides a kit comprisingpackaging material containing: a) a capture moiety that binds to theextracellular domain of the TGF-β Type II receptor; and b) a detectionmoiety capable of binding to an immunoglobulin.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic depicting the different forms of TGF-beta.TGF-beta is produced as a small latent complex (1) which is associatedwith the propeptide region termed LAP (thin black lines). During, orafter secretion, of the small latent complex, additional proteins(hatched oval), e.g., LTBP-1, bind to the small latent complex to formthe large latent complex (2). Latent complexes can be converted to theactive form of TGF-beta, e.g., the 25 kD dimer (5) or the 25 kD dimerwhich is associated with a peptide of LAP (6). Examples of mature formsof TGF-beta are TGF-beta associated with lipoprotein (stippled oval) (3)or TGF-beta associated with a matrix protein (helical fiber) (4), e.g.,fibrillin.

FIG. 2 depicts the association of increasing amounts of lipoprotein with(A) a reduction in TGF-beta binding to the TGF-beta receptor (R2X); and(B) an increasing amount of TGF-beta necessary to half maximally inhibitmink lung cell proliferation.

FIG. 3 depicts the association of TGF-beta with different lipoproteinclasses. Profile of lipoprotein particle elution measured as totalcholesterol ( . . . ) and TGF-beta elution (open circles) followingseparation of the lipoprotein fraction (d<1.215 g/cm³) by gel filtrationchromatography. The position of the major lipoprotein classes are markedby reference to the elution times of the major apolipoproteins. (a)Healthy individual A (b) Healthy individual C (c) Diabetic individual K(d) Diabetic individual L. Letters designating the individuals shownrefer to individuals in Table 1.

FIG. 4 depicts the effect of fish oil therapy on the association ofTGF-beta with lipoprotein. Platelet-poor plasma was prepared from 36individuals prior to receiving fish oil, after 4 weeks of dietarysupplementation with 2.4 g/day fish oil and then after 9 weeks with nofish oil supplementation.

FIG. 5 depicts the effect of aspirin on vascular smooth muscle cells. A)Dose response curve showing the inhibitory effects of increasing amountsof aspirin on human vascular smooth muscle cell proliferation. B)Percent increase in cell number in treated versus untreated humanvascular smooth muscle cells.

FIG. 6 depicts the relationship between TGF-beta concentration found inthe sera of normal individuals (A), individuals with triple vesseldisease (B) and both populations (C), who were undergoing aspirintherapy.

FIG. 7 depicts the effect of tamoxifen (TMX) treatment on plasmaTGF-beta over time. Active TGF-beta () and (a+1) TGF-beta (□) wereassayed by ELISA in platelet poor plasma drawn at various times afterbeginning treatment with 40 mg/day TMX.

FIG. 8 depicts the effect of tamoxifen (TMX) on various cardiovascularrisk factors. A) Lipoprotein(a) amounts. B) Proportion of TGF-betaassociated with the lipoprotein fraction.

FIG. 9 depicts the lesion area in C57B16, apo(a) or apo(E)−/− mice fed anormal diet, high fat diet or high fat diet supplemented with TMX.

FIG. 10 depicts the distribution of TGF-beta between the plasma (opensegment) and various lipoprotein fractions at baseline (a) and after 8hours during a fat tolerance test (b).

FIG. 11 shows the structure of the compounds MER25, zindoxifene, DDAC(Analog II), and DTAC (102b).

FIG. 12 depicts the pathways by which steroid and steroid-mimetic drugsact to produce anti-inflammatory effects and also undesirable sideeffects. The therapeutic action of ER/NFkB modulators is also depicted.

FIG. 13 depicts the pathway by which ER/NFkB modulators upregulatecellular mRNA encoding for TGF-beta.

DETAILED DESCRIPTION OF THE INVENTION

Administration of a Therapeutic Agent

The invention provides a method of treating a mammal having, or at riskof, a indication (e.g. a vascular indication) associated with a TGF-betadeficiency. The invention also provides a method to maintain elevatedlevels of TGF-beta in a mammal which is not imminently at risk of, ordoes not have, an indication associated with a deficiency in TGF-betalevels. The methods comprise the administration of at least onetherapeutic agent that elevates the level of TGF-beta in said mammal.Preferably, the agent elevates the level of latent TGF-beta, for exampleby causing an increase in the level of TGF-beta mRNA, causing anincrease in the translational efficiency of TGF-beta mRNA, or by causingan increase in the secretion of latent TGF-beta.

Another preferred embodiment is an agent that increases the level ofTGF-beta which is capable of binding to the TGF-beta receptors, forexample by causing the release of TGF-beta from matrix components ofplasma proteins, by causing the release of TGF-beta from lipoproteincomplexes, or by causing an increase in the conversion of the latent tothe active form of TGF-beta.

Yet another embodiment of the invention employs the systemicadministration of a therapeutic agent, e.g., a compound of formula (I)including a pharmaceutically acceptable salt thereof, or a combinationthereof, in an amount effective to inhibit or reduce the diminution invessel lumen diameter in a diseased, e.g., atherosclerotic, ortraumatized, e.g., due to stent placement, vessel.

Systemic administration of a therapeutic agent can also be employed totreat or prevent pre-atherosclerotic conditions, e.g., in patients at ahigh risk of developing atherosclerosis or exhibiting signs ofhypertension resulting from atherosclerotic changes in vessels or vesselstenosis due to hypertrophy of the vessel wall. Preferably, thetherapeutic agent is administered orally. It is also preferred that theagent useful in the practice of the invention is administeredcontinually over a preselected period of time or administered in aseries of spaced doses, i.e., intermittently, for a period of time as apreventative measure.

For the prevention of vessel lumen diminution associated with proceduralvascular trauma, the therapeutic agent can be administered before,during or after the procedure, or any combination thereof. For example,for the prevention of restenosis, a series of spaced doses of thetherapeutic agent, optionally, in sustained release dosage form, ispreferably administered before, during and/or after the traumaticprocedure (e.g., angioplasty). The dose may also be delivered locally,via a catheter introduced into the afflicted vessel during theprocedure. After the traumatic procedure is conducted, a series offollow-up doses can be administered systemically over time, preferablyin a sustained release dosage form, for a time sufficient tosubstantially reduce the risk of, or to prevent, restenosis. A preferredtherapeutic protocol duration for this purpose involves administrationfrom about 3 to about 26 weeks after angioplasty.

Combination Therapies

The invention provides combination therapies, i.e., the administrationof at least two therapeutic agents which together are effective tomaintain or elevate TGF-beta levels in a mammal. Accordingly, theinvention provides a method of preventing or treating a mammal having,or at risk of, an indication which is associated with a TGF-betadeficiency, comprising administering an amount of a first agenteffective to elevate the level of latent TGF-beta and an amount of asecond agent effective to increase the level of TGF-beta which iscapable of binding to the TGF-beta receptors, wherein said amounts areeffective to increase the TGF-beta levels in said mammal.

The invention also provides a method comprising administering an amountof a combination of aspirin or an aspirinate and at least one omega-3fatty acid, wherein said amount is effective to maintain or elevate thelevel of TGF-beta in said mammal.

The invention also provides a method of preventing or treating a mammalhaving, or at risk of, a vascular indication which is associated with aTGF-beta deficiency, comprising administering an effective amount of acombination of an aspirinate and at least one omega-3 fatty acid,wherein said amount is effective to increase the level of TGF-beta so asto inhibit or reduce vessel lumen diameter diminution. The inventionalso provides for the administration of at least two therapeutic agentswhich together are effective to elevate the levels of TGF-beta in amammal so as to inhibit or reduce vessel lumen diameter diminution. Theinvention also provides combination therapies to maintain elevatedlevels of TGF-beta in a mammal which is not imminently at risk of, ordoes not have, a vascular indication associated with a deficiency inTGF-beta levels. The therapeutic agents can be selected to act in asynergistic, rather than in an additive, manner to elevate TGF-betalevels. The therapeutic agents can be administered simultaneously as asingle dose, simultaneously in individual doses, or sequentially.

One embodiment of the invention employs the systemic administration of afirst therapeutic agent, e.g., an aspirinate such as copper2-acetylsalicylate, a compound of formula (I), or a combination thereof,in combination with a second therapeutic agent, e.g., a compound offormula (VI), in an amount effective to increase TGF-beta levels. Theincrease in TGF-beta levels, in turn, inhibits or reduces the diminutionin vessel lumen diameter in a diseased, e.g., atherosclerotic, ortraumatized, e.g., due to stent placement, vessel. The increase inTGF-beta levels can also inhibit atherosclerotic lesion formation ordevelopment, increase plaque stability and/or promote lesion regression.

Systemic administration of the therapeutic agents can also be employedto treat or prevent pre-atherosclerotic conditions, e.g., in patients ata high risk of developing atherosclerosis or exhibiting signs ofhypertension resulting from atherosclerotic changes in vessels or vesselstenosis due to hypertrophy of the vessel wall. Preferably, at least oneof the therapeutic agents is administered orally.

It is also preferred that the agents useful in the practice of theinvention are administered continually over a preselected period of timeor administered in a series of spaced doses, i.e., intermittently, for aperiod of time as a preventative measure.

A preferred embodiment of the invention provides a method for thetreatment or prevention of atherosclerosis, wherein an omega-3 fattyacid in combination with aspirin or an aspirinate, is administered so asto inhibit (block or reduce) atherosclerotic lesion formation ordevelopment, e.g., so as to inhibit lipid accumulation, increase plaquestability or promote lesion regression. In this embodiment of theinvention, it is preferred that the therapeutic agents are orallyadministered. Preferably, copper aspirinate and an omega-3 fatty acidare orally administered. A preferred source of the omega-3 fatty acid isfish oil.

Another preferred embodiment of the invention provides a method for thetreatment or prevention of atherosclerosis, wherein at least twotherapeutic agents of the invention are administered in combination soas to inhibit (block or reduce) atherosclerotic lesion formation ordevelopment, e.g., so as to inhibit lipid accumulation, increase plaquestability or promote lesion regression. In this embodiment of theinvention, it is preferred that at least one of the therapeutic agentsis orally administered.

Combination therapies are also useful to treat vessels traumatized byinterventional procedures. For example, for the prevention ofrestenosis, a series of spaced doses of at least two of the presenttherapeutic agents, optionally, in sustained release dosage form, arepreferably administered before and after the traumatic procedure (e.g.,angioplasty). The dose may also be delivered locally, via a catheterintroduced into the afflicted vessel during the procedure. After theprocedure is conducted, a series of follow-up doses of, optionally, bothagents, can be administered systemically, preferably in a sustainedrelease dosage form, for a time sufficient to substantially reduce therisk of, or to prevent, restenosis. As noted above, a preferred durationfor this purpose is from about 3 to about 26 weeks after angioplasty.

Kits Comprising a Delivery Device and the Therapeutic Agents of theInvention

The invention provides a kit comprising packing material enclosing,separately packaged, at least one device adapted for the local orsystemic delivery of a therapeutic agent, e.g., a catheter, a valve, astent, a stet, a shunt or a synthetic graft, and at least one unitdosage form, as well as instruction means for their use, in accord withthe present methods. A valve, stent or shunt useful in the methods ofthe invention can comprise a biodegradable coating or porousnon-biodegradable coating, having dispersed therein a therapeutic agentof the invention, preferably a sustained release dosage form of thetherapeutic agent. The unit dosage form comprises an amount of at leastone of the present therapeutic agents effective to accomplish thetherapeutic results described herein when delivered locally and/orsystemically. A preferred embodiment of the invention is a kitcomprising a catheter adapted for the local delivery of at least onetherapeutic agent to a site in the lumen of a mammalian vessel, alongwith instruction means directing its use in accord with the presentinvention. Preferably, the therapeutic agent comprises a copperaspirinate.

The invention provides a kit comprising packing material enclosing,separately packaged, at least one device adapted for the local orsystemic delivery of a therapeutic agent, e.g., a catheter, a valve, astent, a stet, a shunt or a synthetic graft, and at least one unitdosage form which may comprise an amount of at least two of the presenttherapeutic agents effective to accomplish the therapeutic resultsdescribed herein.

Another embodiment of the invention is a kit comprising a catheteradapted for the local delivery of at least two therapeutic agents, aunit dosage of a first therapeutic agent, and a unit dosage of a secondtherapeutic agent, along with instruction means directing their use inaccord with the present invention. The unit dosage forms of the firstand second agents may be introduced via discrete lumens of a catheter,or mixed together prior to introduction into a single lumen of acatheter. If the unit dosage forms are introduced into discrete lumensof a catheter, the delivery of the agents to the vessel can occursimultaneously or sequentially. Moreover, a single lumen catheter may beemployed to deliver a unit dosage form of one agent, followed by thereloading of the lumen with another agent and delivery of the otheragent to the lumen of the vessel. Either or both unit dosages can act toreduce the diminution in vessel lumen diameter at the target site.

Alternatively, a unit dosage of one of the therapeutic agents may beadministered locally, e.g., via catheter, while a unit dosage of anothertherapeutic agent is administered systemically, e.g., via oraladministration. It is also envisioned that the kit of the inventioncomprises a non-catheter delivery device, e.g., a valve, stet, stent orshunt, for systemic or local delivery of a compound of formula (I-VI). Avalve, stent or shunt useful in the methods of the invention cancomprise a biodegradable coating or porous non-biodegradable coating,having dispersed therein one or more therapeutic agents of theinvention, preferably a sustained release dosage form of the therapeuticagent.

Definitions

The following definitions apply.

“Abnormal or pathological or inappropriate” with respect to an activityor proliferation means division, growth or migration of normal cells,but not cancerous or neoplastic cells, occurring more rapidly or to asignificantly greater extent than typically occurs in a normallyfunctioning cell of the same type, or in lesions not found in healthytissues.

“Agents which activate the latent form of TGF-beta to the active form”include, but are not limited to, moieties such as hydrocortisone,dexamethasone, a compound of formula (VI) (such as tamoxifen), VitaminD3 and retinoic acid (vitamin A); plasmin stimulators, e.g., Lp(a)lowering agents such as tamoxifen, PAI-1 lowering agents (e.g.,simvastatin and other VLDL-lowering agents), and agents which exhibitincreased tPA activity (e.g., retinoids, such as Vitamin D3); and agentswhich exhibit non-plasmin mediated activation (e.g., thrombospondin andVitamin D3).

“Agents which increase the level of TGF-beta which is capable of bindingto the TGF-beta receptors” includes moieties capable of activating thelatent form of TGF-beta to the active form thereof, moieties whichrelease TGF-beta from complexes of matrix components and TGF-beta,complexes of plasma proteins and TGF-beta and/or complexes oflipoproteins and TGF-beta. A number of compounds of formula (VI) canincrease the level of TGF-beta which is capable of binding to theTGF-beta receptors.

“Agents which release TGF-beta from the extracellular matrix” includemoieties such as heparin, heparin sugar analogs (e.g., fucoidin) andbetaglycan proteoglycan chains.

“Agents which release TGF-beta from lipoprotein sequestration” includemoieties such as Vitamin E and its salts (e.g., Vitamin E succinate),fish oil, simvastatin, other VLDL-lowering agents, apo-AII-loweringagents, and apoAI-stimulating agents.

“ApoAII-lowering agent” includes an agent which decreases the synthesisof apoAII, decreases the post-translational insertion of apoAII intonascent HDL particles or stimulates the clearance of apoAII-containingparticles, e.g., by immunoapheresis of plasma with anti-apoAIIantibodies.

“ApoAI-stimulating agent” includes an agent which stimulates thesynthesis of apoAI, stimulates HDL production or extends the half-lifeof apoAI-HDL particles. For example, estrogen or estrogen agonists, oranalogs and derivatives thereof, an agonist of hepatic nuclear factor(HNF) 3 or 4, or an agonist of the retinoid receptor, may increase apoAItranscription.

“Aspirinate” refers generally to aspirin derivatives and analogs,including pharmaceutically acceptable salts thereof, with the exceptionthat aspirin itself is not included within the term “aspirinate”. Theterm includes, but is not limited to, 3,5-diisopropyl salicylic acid,salicylic acid, 3,5-di(tertiarybutyl)salicylic acid, adamantylsalicylicacid, 3,5-dibromoacetylsalicylic acid, 3,5-diiodoacetylsalicylic acid,4-(tertiarybutyl)salicylic acid, 4-nitrosalicylic acid, 4-aminosalicylicacid, 4-acetylaminosalicylic acid, 5-chlorosalicylic acid,3,5-dichlorosalicylic acid and salts thereof, and compounds of formula(I) and their salts. Preferably, the aspirinate is provided inessentially pure form, most preferably in a unit dosage form, incombination with one or more pharmaceutically acceptable carriers,including vehicles and/or excipients. Preferably, the aspirinate is in aform suitable for oral administration, and more preferably theaspirinate is in combination with a liquid vehicle.

“At least one”, when used with respect to omega-3 fatty acids would berecognized in the art as indicating that a plurality, about 1 to 30,preferably about 1 to 25, more preferably about 2 to 20, of omega-3fatty acids are often present in natural sources of these compounds.

“Autoimmune disease” means a disease which is characterized by thepresence of autoreactive T lymphocytes resulting in pathologicalinflammation and subsequent damage or destruction of the target tissue.Such diseases include, but are not limited to, rheumatoid arthritis,multiple sclerosis and late-onset diabetes.

“Betaglycan proteoglycan chain” includes all or a portion of any of theproteoglycan that comprise the class of molecules termed type-IIITGF-beta receptor, e.g., CD105, endoglin or betaglycan. For example, aportion of the proteoglycan may include all or a portion of the proteinmoiety of the proteoglycan, all or a portion of the polysaccharidemoiety of the proteoglycan, all or a portion of the protein moiety and aportion of the polysaccharide moiety, all or a portion of thepolysaccharide moiety and a portion of the protein moiety, or a portionof the protein moiety and a portion of the polysaccharide moiety.Preferably, the betaglycan proteoglycan chain has a similar or greateraffinity for TGF-beta relative to the affinity of native betaglycan forTGF-beta.

“Bioavailable” TGF-beta means TGF-beta which is in a form capable ofbinding to the TGF-beta receptors, i.e., eliciting a biological effect.For example, TGF-beta which is in a complex with matrix components orplasma proteins, or lipoproteins, is generally not “bioavailable” or hasreduced bioavailability relative to TGF-beta which is not complexed withmatrix components, plasma proteins, or lipoproteins.

“Cholesterol lowering agents” include agents which are useful forlowering serum cholesterol such as for example bile acid sequesteringresins (e.g. colestipol hydrochloride or cholestyramine), fibric acidderivatives (e.g. clofibrate, fenofibrate, or gemfibrozil),thiazolidenediones (e.g. troglitazone), or HMG-CoA reductase inhibitors(e.g. fluvastatin sodium, lovastatin, pravastatin sodium, orsimvastatin), as well as nicotinic acid, niacin, or probucol.

“Elevated” TGF-beta levels means that the TGF-beta levels in vivo aregreater after administration of the therapeutic agent than beforeadministration. Thus, for example, active TGF-beta levels may beincreased after administration, but may be less than normal levels,similar to normal levels or greater than normal levels of TGF-beta invivo.

“Heparin sugar analogs” includes any sulfated polysaccharide which is acomponent of heparin sulfate proteoglycan, or a sulfated polysaccharidehaving a structure similar to the polysaccharide chain of heparinsulphate proteoglycan:

“NFkB” means any of the family of transcription factor complexes whichhave as at least one of their components the subunits known as p65(Re1A), p50, p52, c-re1, p68 (Re1B) as well as the complexes which haveas at least one of their components the endogenous inhibitors of NFkBactivity, known as IkB-alpha, MAD3, pp40, IkB-beta and IkB-gamma as wellas their functional equivalents, analogs and derivatives thereof.

“NFkB activity” means activation of genes associated with theinflammatory state resulting from direct binding of an NFkBtranscription factor complex to DNA elements, including, but not limitedto, the kB element in the immunoglobulin kappa light chain gene. NFkBcomplex is normally retained in the cytoplasm by interaction with itsendogenous inhibitor IkB. NFkB activity must be preceded by localizationof the NFkB complex to the nucleus. However, translocation of the NFkBcomplex to the nucleus does not constitute NFkB activity unlesstranscription from genes associated with the inflammatory state isstimulated.

“Non-vascular indication” means diseases and conditions which areassociated with TGF-beta deficiency, other than those diseases andconditions defined herein as vascular indications. Non-vascularindications include, but is not limited to cancer, Marfan's syndrome,Parkinson's disease, fibrosis, Alzheimer's disease, senile dementia,osteoporosis, diseases associated with inflammation, such as rheumatoidarthritis, multiple sclerosis and lupus erythematosus, as well as otherauto-immune disorders. Non-vascular indications also include thepromotion of wound healing and the lowering of serum cholesterol levels.

“Omega-3 fatty acid” includes synthetic or naturally occurring sourcesof omega-3 fatty acids, such as fish oil, e.g., cod liver oil, walnutsand walnut oil, wheat germ oil, rapeseed oil, soybean lecithin,soybeans, tofu, common beans, butternuts, seaweed and flax seed oil. Theomega-3 fatty acids include (C₁₆-C₂₄) alkanoic acids comprising 5-7double bonds, wherein the last double bond is located between the thirdand fourth carbon atom from the methyl end of the fatty acid chain.These fatty acids have been proposed to yield significant cardiovascularprotection (Burr et al., Lancet, 221 757 (1989)). Omega-3 fatty acidsinclude 5, 8, 11, 14, 17-eicosapentaenoic acid and docosahexaenoic acid.See The Merck Index (11th ed. 1989) at entry 3495, and references citedtherein.

“Pathological inflammation” means an increase in the recruitment andactivation of immune cells, or residence and activation of immune cellsfor a longer period of time, in a particular tissue or tissues in anindividual relative to an individual not at risk or, or afflicted with,an autoimmune disease. For the purposes of this description, theprototypical cells upon which the effects of ER/NFkB modulators arefelt, are cells of the immune system, including but limited to,autoreactive T lymphocytes, alloreactive T lymphocytes, B lymphocytes,monocytes, tissue macrophages, neutrophils, eosinophils and otherleukocytes. However, the usefulness of ER/NFkB modulators is not limitedto their effects on immune cells in the treatment of autoimmunediseases. Effects on vascular endothelial cells and on the cellscomposing the target tissue may also contribute to the anti-inflammatoryeffect of the ER/NFkB modulators by reducing recruitment of leukocytesas well as activation of resident immune cells.

“PAI-1 lowering agent” includes an agent which increases insulinsensitivity, decreases production of PAI-1 or decreases the activity ofPAI-1 as an inhibitor of plasminogen activators or of plasmin. PAI-1lowering agent includes the thiazolidenediones (e.g. troglitazone).

“Plasmin stimulator” includes an agent which increases the activity ofplasmin, e.g., a PAI-1 inhibitor, tissue plasminogen activator (tPA) orstreptokinase, preferably without disrupting normal hemostasis. Aplasmin stimulator may increase plasmin levels by catalyzing theconversion of the latent form of plasmin, i.e., plasminogen, to theactive form, or stimulate the activity of the plasmin enzyme, e.g.,generally or with regard to a specific substrate, e.g., TGF-beta.

“Procedural vascular trauma” includes the effects ofsurgical/medical-mechanical interventions into mammalian vasculature,but does not include vascular trauma due to the organic vascularpathologies listed hereinabove, or to unintended traumas, such as due toan accident. Thus, procedural vascular traumas within the scope of thepresent treatment method include (1) organ grafting or transplantation,such as transplantation and grafting of heart, kidney, liver and thelike, e.g., involving vessel anastomosis; (2) vascular surgery, such ascoronary bypass surgery, biopsy, heart valve replacement, atheroectomy,thrombectomy, and the like; (3) transcatheter vascular therapies (TVT)including angioplasty, e.g., laser angioplasty and PTCA proceduresdiscussed hereinbelow, employing balloon catheters, or indwellingcatheters; (4) vascular grafting using natural or synthetic materials,such as in saphenous vein coronary bypass grafts, dacron and venousgrafts used for peripheral arterial reconstruction, etc.; (5) placementof a mechanical shunt, such as a PTFE hemodialysis shunt used forarteriovenous communications; and (6) placement of an intravascularstent, which may be metallic, plastic or a biodegradable polymer. SeeU.S. patent application Ser. No. 08/389,712, filed Feb. 15, 1995, whichis incorporated by reference herein. For a general discussion ofimplantable devices and biomaterials from which they can be formed, seeH. Kambic et al., “Biomaterials in Artificial Organs”, Chem. Eng. News,30 (Apr. 14, 1986), the disclosure of which is incorporated by referenceherein.

“Proliferation,” means an increase in cell number, i.e., by mitosis ofthe cells.

“Sustained release” means a dosage form designed to release atherapeutic agent therefrom for a time period ranging from at leastabout 0.0005 to about 21, and more preferably at least about 1-3 toabout 120, days. Release over a longer time period is also contemplatedas “sustained release” in the context of the dosage form of the presentinvention. It is contemplated that sustained release dosage forms forsystemic administration as well as local administration can be employedin the practice of the invention. Examples of sustained release dosageforms are disclosed in co-pending application Ser. No. 08/478,936, filedJun. 7, 1995, the disclosure of which is incorporated by referenceherein.

“Tamoxifen” includestrans-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine, andthe pharmaceutically acceptable salts thereof, which are capable ofenhancing the level of active TGF-beta, e.g., by increasing the level oflatent TGF-beta or by increasing the level of TGF-beta which is capableof binding to the TGF-beta receptors.

“TGF-beta” includes transforming growth factor-beta as well asfunctional equivalents, derivatives and analogs thereof, e.g., TGF-β₁,TGF-β₂, and TGF-β₃. The TGF-beta isoforms are a family ofmultifunctional, disulfide-linked dimeric polypeptides that affectactivity, proliferation and differentiation of various cells types. Afunctional equivalent of TGF-β can include agents that bind to the TGF-βreceptor, e.g. a receptor agonist or antagonist or a neutral bindingagent, and/or which induces the same biological response as TGF-β.

“Vascular indication” includes, but is not limited to, a cardiovasculardisease, e.g., atherosclerosis, thrombosis, myocardial infarction, andstroke, or a cardiovascular condition, e.g., vessels subjected to traumaassociated with interventional procedures (“procedural vasculartrauma”), such as restenosis following angioplasty, placement of ashunt, stet, stent, synthetic or natural excision grafts, indwellingcatheter, valve or other implantable devices. Also within the scope ofthe term “vascular indication” is non-coronary vessel disease, such asarteriolosclerosis, small vessel disease, nephropathy, greater thannormal levels of serum cholesterol, asthma, hypertension, emphysema andchronic obstructive pulmonary disease. “Vascular indication” does notinclude cancer, including smooth muscle cell carcinomas or neoplasms, oridiopathic symptoms such as forms of angina that are not attributable tovascular diseases.

Small vessel disease includes, but is not limited to, vascularinsufficiency in the limbs, peripheral neuropathy and retinopathy, e.g.,diabetic retinopathy. “VLDL-lowering agent” includes an agent whichdecreases the hepatic synthesis of triglyceride-rich lipoproteins orincreases the catabolism of triglyceride-rich lipoproteins, e.g.,fibrates such as gemfibrozil, or the statins, increases the expressionof the apoE-mediated clearance pathway, or improves insulin sensitivityin diabetics, e.g., the thiazolidene diones.

Additionally, as used herein, “agents which increase the level of latentTGF-beta” include moieties capable of stimulating the production ofTGF-beta protein (generally the latent form thereof). The mechanismleading to the increase in TGF-beta protein can include, but is notlimited to, up-regulation of mRNA production (transcription), increasedtranslational efficiency of the mRNA, or increased secretion of thelatent TGF-beta complex. Agents which increase the production ofTGF-beta protein include, but are not limited to, moieties which affectthe nuclear hormone receptor pathway (e.g., tamoxifen, idoxifene,toremifene, raloxifene, droloxifene and other anti-estrogen analogues oftamoxifen, ethynyl estradiol, diethylstilbestrol, other syntheticestrogen agonists and compounds disclosed in U.S. Pat. Nos. 4,442,119,5,015,666, 5,098,903, 5,324,736), 1,25 dihydroxy-vitamin D3,allopurinol, EB 1089, MC 903, KH1060, retinoic acid/vitamin A and ligandpharmaceutical analogs thereof (Mukherjee et al. Nature, 1997, 386:407-410), dexamethasone (e.g., glucocorticoid agonist analogues),progesterone (e.g., gestodene and synthetic progestins), and thyroidhormone analogues (e.g. sodium liothyronine and sodium levothyroxine),(e.g. 12,14 dideoxy-prostaglandin J2; Δ12,14-PGJ2).

Other agents which increase the level of TGF-beta include aspirin,aspirinates such as copper aspirinate, and red wine extract (see ExampleIV). Red wine extract is a fraction or concentrate derived from red winethat is substantially enriched in copper aspirinate, hexamethylenebisacetamide, 4-hydroxyquinazoline, coumarin and benzocaine.

The term “halo” includes fluoro, chloro, bromo, or iodo. The termsalkyl, and alkoxy denote both straight and branched groups; butreference to an individual radical such as “propyl” embraces only thestraight chain radical, a branched chain isomer such as “isopropyl”being specifically referred to. Aryl denotes a phenyl radical or anortho-fused bicyclic carbocyclic radical having about nine to ten ringatoms in which at least one ring is aromatic. Heteroaryl encompasses aradical attached via a ring carbon of a monocyclic aromatic ringcontaining five or six ring atoms consisting of carbon and one to fourheteroatoms each selected from the group consisting of non-peroxideoxygen, sulfur, and N(X) wherein X is absent or is hydrogen, O,(C₁-C₄)alkyl, phenyl or benzyl, as well as a radical of an ortho-fusedbicyclic heterocycle of about eight to ten ring atoms derived therefrom,particularly a benz-derivative or one derived by fusing a propylene,trimethylene, or tetramethylene diradical thereto.

It will be appreciated by those skilled in the art that compounds of theinvention having a chiral center may exist in and be isolated inoptically active and racemic forms. Some compounds may exhibitpolymorphism. It is to be understood that the present inventionencompasses any racemic, optically-active, polymorphic, orstereoisomeric form, or mixtures thereof, of a compound of theinvention, which possess the useful properties described herein, itbeing well known in the art how to prepare optically active forms (forexample, by resolution of the racemic form by recrystallizationtechniques, by synthesis from optically-active starting materials, bychiral synthesis, or by chromatographic separation using a chiralstationary phase).

Specific values listed for radicals, substituents, and ranges, are forillustration only and they do not exclude other defined values or othervalues within defined ranges for the radicals and substituents.

Specifically, (C₁-C₃)alkyl can be methyl, ethyl, propyl, or isopropyl;(C₁-C₄)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butylor sec-butyl; (C₁-C₆)alkyl can be methyl, ethyl, propyl, isopropyl,butyl, iso-butyl, sec-butyl, pentyl, isopentyl, neopentyl, or hexyl;(C₁-C₁₂)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl,sec-butyl, pentyl, isopentyl, neo-pentyl, hexyl, 2-hexyl, 3-hexyl,heptyl, 2-heptyl, 3-heptyl, octyl, 2-octyl, 3-octyl, 4-octyl, nonyl,2-nonyl, 3-nonyl, 4-nonyl, decyl, 2-decyl, 3-decyl, 4-decyl, 5-decyl,undecyl, 2-undecyl, 3-undecyl, 4-undecyl, 5-undecyl, dodecyl, 2-dodecyl,3-dodecyl, 4-dodecyl, 5-dodecyl, or 6-dodecyl; (C₃-C₆)cycloalkyl can becyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl; (C₃-C₆)cycloalkenylcan be cyclopropenyl, cyclobutenyl, cyclopentenyl, cyclopentadienylcyclohexenyl, or cyclohexadienyl; (C₁-C₆)alkoxy can be methoxy, ethoxy,propoxy, isopropoxy, butoxy, iso-butoxy, sec-butoxy, pentoxy,neopentoxy, isopentoxy, or hexoxy; (C₁-C₆)alkanoyl can be acetyl,propanoyl or butanoyl; (C₂-C₆)alkanoyloxy can be acetoxy, propanoyloxyor butanoyloxy; halo(C₁-C₁₂)alkyl can be fluoromethyl, difluoromethyl,trifluoromethyl, fluoroethyl, difluoroethyl, trifluoroethyl,perfluoroethyl, fluoropropyl, difluoropropyl, trifluoropropyl,fluorobutyl, difluorobutyl, trifluorobutyl, fluoropentyl,difluoropentyl, trifluoropentyl, fluorohexyl, difluorohexyl,trifluorohexyl, chloroethyl, dichloroethyl, trichloroethyl,perchloroethyl, chloropropyl, dichloropropyl, trichloropropyl,chlorobutyl, dichlorobutyl, trichlorobutyl, chloropentyl,dichloropentyl, trichloropentyl, chlorohexyl, dichlorohexyl,trichlorohexyl, bromoethyl, dibromoethyl, tribromoethyl, perbromoethyl,bromopropyl, dibromopropyl, tribromopropyl, bromobutyl, dibromobutyl,tribromobutyl, bromopentyl, dibromopentyl, tribromopentyl, bromohexyl,dibromohexyl, tribromohexyl, iodoethyl, iodopropyl, iodobutyl,iodopentyl, iodohexyl, haloheptyl, dihaloheptyl, trihaloheptyl,halooctyl, dihalooctyl, trihalooctyl, halononyl, dihalononyl,trihalononyl, halodecyl, dihalodecyl, trihalodecyl, haloundecyl,dihaloundecyl, trihaloundecyl, halododecyl, dihalododecyl, ortrihalododecyl.

Likewise, aryl can be phenyl, indenyl, or naphthyl; heteroaryl can befuryl, imidazolyl, tetrazolyl, pyridyl, (or its N-oxide), thienyl,pyrimidinyl (or its N-oxide), indolyl, or quinolyl (or its N-oxide); andaryl (C₁-C₃)alkyl can be benzyl, indenylmethyl, naphthylmethyl,phenethyl, indenylethyl, naphthylethyl, phenylpropyl, indenylpropyl, ornaphthylpropyl; and heteroaryl (C₁-C₃)alkyl can be furylmethyl,imidazolylmethyl, tetrazolylmethyl, pyridylmethyl (or its N-oxide),thienyhnethyl, pyrimidinylmethyl (or its N-oxide), indolylmethyl,quinolylmethyl, furylethyl, imidazolylethyl, tetrazolylethyl,pyridylethyl, (or its N-oxide), thienylethyl, pyrimidinylethyl (or itsN-oxide), indolylethyl, quinolylethyl, furylpropyl, imidazolylpropyl,tetrazolylpropyl, pyridylpropyl, (or its N-oxide), thienylpropyl,pyrimidinylpropyl (or its N-oxide), indolylpropyl, or quinolylpropyl.

More specifically, (C₁-C₃)alkyl can be methyl, ethyl, or propyl;(C₁-C₄)alkyl can be methyl, ethyl, propyl, or butyl; (C₁-C₆)alkyl can bemethyl, ethyl, propyl, isopropyl, butyl, iso-butyl, pentyl, or hexyl;(C₁-C₁₂)alkyl can be methyl, ethyl, propyl, isopropyl, butyl, iso-butyl,sec-butyl, pentyl, hexyl, heptyl, or octyl; (C₃-C₆)cycloalkyl can becyclopentyl, or cyclohexyl; (C₃-C₆)cycloalkenyl can be 2-cyclopentenyl,3-cyclopentenyl, 2-cyclohexenyl, or 3-cyclohexenyl; (C₁-C₆)alkoxy can bemethoxy, ethoxy, or propoxy; (C₁-C₆)alkanoyl can be acetyl;(C₂-C₆)alkanoyloxy can be acetoxy; halo(C₁-C₁₂)alkyl can befluoromethyl, difluoromethyl, trifluoromethyl, trifluoroethyl,fluoropropyl, trifluoropropyl, fluorobutyl, trifluorobutyl,fluoropentyl, trifluoropentyl, fluorohexyl, trifluorohexyl, chloroethyl,chloropropyl, chlorobutyl, bromoethyl, bromopropyl, bromobutyl,iodoethyl, iodopropyl, iodobutyl; aryl can be phenyl, heteroaryl can befuryl, imidazolyl, pyridyl (or its N-oxide), or thienyl; aryl(C₁-C₃)alkyl can be benzyl or phenethyl; and heteroaryl (C₁-C₃)alkyl canbe furylmethyl, imidazolylmethyl, pyridylmethyl (or its N-oxide), orthienylmethyl.

Compounds of Formula (I) Within the Scope of the Invention

A specific aspirinate useful in the present invention is a compound offormula (I):

wherein

R¹ is hydrogen, halo, nitro, cyano, hydroxy, CF₃, —NR_(c)R_(d),—C(═O)OR_(e), —OC(═O)OR_(e), —C(═N)OR_(e), (C₁-C₆)alkyl or(C₁-C₆)alkoxy;

R² is hydrogen or —XR_(a);

R³ is —C(═O)YR_(b);

R⁴ is (═O)_(n); or R⁴ is (C₁-C₆)alkyl, (C₁-C₆)alkanoyl or(C₂-C₆)alkanoyloxy and forms a sulfonium salt with the thiophene sulfur,wherein the associated counter ion is a pharmaceutically acceptableanion;

R⁵ is hydrogen;

n is 0, 1 or 2;

X is oxygen or sulfur;

Y is oxygen or sulfur;

R_(a) is (C₁-C₆)alkanoyl;

R_(b) is hydrogen or (C₁-C₃)alkyl;

R_(c) and R_(d) are each independently hydrogen, (C₁-C₄)alkyl, phenyl,C(═O)OH, C(═O)O(C₁-C₄)alkyl CH₂C(═O)OH, CH₂C(═O)O(C₁-C₄)alkyl, or(C₁-C₄)alkoxy; or R_(c) and R_(d) together with the nitrogen to whichthey are attached are a 3, 4, 5, or 6 membered heterocyclic ring; and

R_(e) is hydrogen or (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt thereof;

provided that R² and R³ are on adjacent positions of the ring to whichthey are attached, or are on the 2- and 5-positions of the ring; andfurther provided that when R² is hydrogen; R³ is on the 2-or 5-positionof the ring to which it is attached and R⁴ is (C₁-C₄)alkanoyloxy.

A specific aspirinate of formula I useful in the present invention is acompound of formula (II):

wherein X is O or S; Y is O or S; R¹ is hydrogen, halo, nitro, cyano,hydroxy, CF₃, —NR_(c)R_(d), —C(═O)OR_(e), —OC(═O)OR_(e), —OC(═N)OR_(e),(C₁-C₆)alkyl or (C₁-C₆)alkoxy; and R_(c) and R_(d) are eachindependently hydrogen, (C₁-C₄)alkyl, phenyl, —C(═O)OH,—C(═O)O(C₁-C₄)alkyl, —CH₂C(═O)OH, —CH₂C(═O)O(C₁-C₄)alkyl, or(C₁-C₄)alkoxy; or R_(c) and R_(d) together with the nitrogen to whichthey are attached are a 3, 4, 5, or 6 membered heterocyclic ring; or apharmaceutically acceptable salt thereof.

A specific aspirinate of formula I useful in the present invention is acompound of formula (III):

wherein X is O or S; Y is O or S; R¹ is hydrogen, halo, nitro, cyano,hydroxy, CF₃, —NR_(c)R_(d), —C(═O)OR_(e), —OC(═O)OR_(e), —C(═N)OR_(e),(C₁-C₆)alkyl or (C₁-C₆)alkoxy; R_(c) and R_(d) are each independentlyhydrogen, (C₁-C₄)alkyl, phenyl, —C(═O)OH, —C(═O)O(C₁-C₄)alkyl,—CH₂C(═O)OH, —CH₂C(═O)O(C₁-C₄)alkyl, or (C₁-C₄)alkoxy; or R_(c) andR_(d) together with the nitrogen to which they are attached are a 3, 4,5, or 6 membered heterocyclic ring; or a pharmaceutically acceptablesalt thereof.

Another specific aspirinate of formula I useful in the present inventionis a compound of formula (IV):

wherein X is O or S; Y is O or S; R¹ is hydrogen, halo, nitro, cyano,hydroxy, CF₃, —NR_(c)R_(d), —C(═O)OR_(e), —OC(═O)OR_(e), —C(═N)OR_(e),(C₁-C₆)alkyl or (C₁-C₆)alkoxy; R_(c) and R_(d) are each independentlyhydrogen, (C₁-C₄)alkyl, phenyl, —C(═O)OH, —C(═O)O(C₁-C₄)alkyl,—CH₂C(═O)OH, —CH₂C(═O)O(C₁-C₄)alkyl, or (C₁-C₄)alkoxy; or R_(c) andR_(d) together with the nitrogen to which they are attached are a 3, 4,5, or 6 membered heterocyclic ring; or a pharmaceutically acceptablesalt thereof.

Another specific aspirinate of formula I useful in the present inventionis a compound of formula (V):

wherein X is O or S; Y is O or S; R¹ is hydrogen, nitro, halo, cyano,hydroxy, or N(R)₂, wherein each R is hydrogen, (C₁-C₄)alkyl, phenyl,COOH, CO₂(C₁-C₄)alkyl, or O[(C₁-C₄)alkyl], R_(c) and R_(d) are eachindependently hydrogen, (C₁-C₄)alkyl, phenyl, —C(═O)OH,—C(═O)O(C₁-C₄)alkyl, —CH₂C(═O)OH, —CH₂C(═O)O(C₁-C₄)alkyl, or(C₁-C₄)alkoxy; or R_(c) and R_(d) together with the nitrogen to whichthey are attached are a 3, 4, 5, or 6 membered heterocyclic ring; or apharmaceutically acceptable salt thereof.

A specific aspirinate useful in the present invention is a compound offormula (I):

wherein

R¹ is hydrogen, halo, nitro, cyano, hydroxy, CF₃, —NR_(c)R_(d),—C(═O)OR_(e), (C₁-C₆)alkyl or (C₁-C₆)alkoxy;

R² is hydrogen or —XR_(a);

R³ is —C(═O)YR_(b);

R⁴ is (═O)_(n); or R⁴ is (C₁C₆)alkyl, (C₁-C₆)alkanoyl or(C₂-C₆)alkanoyloxy and forms a sulfonium salt with the thiophene sulfur,wherein the associated counter ion is a pharmaceutically acceptableanion;

R⁵ is hydrogen;

n is 0, 1 or 2;

X is oxygen or sulfur;

Y is oxygen or sulfur;

R_(a) is (C₁-C₆)alkanoyl;

R_(b) is hydrogen or (C₁-C₃)alkyl;

R_(c) and R_(d) are each independently hydrogen, (C₁-C₄)alkyl, phenyl,COOH, CO₂(C₁-C₄)alkyl or O[(C₁-C₄)alkyl]; or R_(c) and R_(d) togetherwith the nitrogen to which they are attached are pyrrolidino,piperidino, piperazin-1-ly or morpholino; and

R_(e) is hydrogen or (C₁-C₆)alkyl;

or a pharmaceutically acceptable salt thereof;

provided that R² and R³ are on adjacent positions of the ring to whichthey are attached, or are on the 2- and 5-positions of the ring; andfurther provided that when R² is hydrogen; R³ is on the 2-or 5-positionof the ring to which it is attached and R⁴ is (C₁-C₄)alkanoyloxy.

A specific aspirinate useful in the present invention is a compound offormula (I) which is not 3-acetoxy-2-carboxythiophene.

Another specific aspirinate useful in the present invention is acompound of formula (I) wherein R¹ is halo, nitro, cyano, CF₃ or—C(═O)OR_(e); or a pharmaceutically acceptable salt thereof.

Yet another specific aspirinate useful in the present invention is acompound of formula (I) wherein R¹ is hydrogen.

A further specific aspirinate useful in the present invention is acompound of formula (I) wherein R² is —XR_(a).

A specific aspirinate useful in the present invention is a compound offormula (I) wherein R⁴ is (C₁-C₆)alkyl, (C₁-C₆)alkanoyl or(C₂-C₆)alkanoyloxy and forms a sulfonium salt with the thiophene sulfur,wherein the associated counter ion is a pharmaceutically acceptableanion.

A specific aspirinate useful in the present invention is a compound offormula (I) wherein R⁵ is hydrogen.

Another specific aspirinate useful in the present invention is acompound of formula (I) wherein R² is in the 3-position, R³ is in the4-position and R¹ is halo, nitro, cyano, hydroxy, CF₃, —NR_(c)R_(d),—C(═O)OR_(e), (C₁-C₆)alkyl, or (C₁-C₆)alkoxy; or a pharmaceuticallyacceptable salt thereof.

Yet another specific aspirinate useful in the present invention is acompound of formula (I) wherein R² is in the 2-position and R³ is in the3-position; and R¹ is halo, nitro, cyano, hydroxy, CF₃, —NR_(c)R_(d),—C(═O)OR_(e), (C₁-C₆)alkyl, or (C₁-C₆)alkoxy; or a pharmaceuticallyacceptable salt thereof.

A specific aspirinate of formula I useful in the present invention is acompound of formula (II):

wherein X is O or S; Y is O or S; R¹ is hydrogen, nitro, halo, cyano,hydroxy, or N(R)₂, wherein each R is hydrogen, (C₁-C₄)alkyl, phenyl,COOH, CO₂(C₁-C₄)alkyl, or O[(C₁-C₄)alkyl]; or a pharmaceuticallyacceptable salt thereof.

A specific aspirinate of formula I useful in the present invention is acompound of formula II wherein, if X═Y═O, then R¹ is not H.

A specific aspirinate of formula I useful in the present invention is acompound of formula (III):

wherein X is O or S; Y is O or S; R¹ is hydrogen, nitro, halo, cyano,hydroxy, or N(R)₂, wherein each R is hydrogen, (C₁-C₄)alkyl, phenyl,COOH, CO₂(C₁-C₄)alkyl, or O[(C₁-C₄)alkyl]; or a pharmaceuticallyacceptable salt thereof.

Another specific aspirinate of formula I useful in the present inventionis a compound of formula (IV):

wherein X is O or S; Y is O or S; R¹ is hydrogen, nitro, halo, cyano,hydroxy, or N(R)₂, wherein each R is hydrogen, (C₁-C₄)alkyl, phenyl,COOH, CO₂(C₁-C₄)alkyl, or O[(C₁-C₄)alkyl], or a pharmaceuticallyacceptable salt thereof.

Another specific aspirinate of formula I useful in the present inventionis a compound of formula (V):

wherein X is O or S; Y is O or S; R¹ is hydrogen, nitro, halo, cyano,hydroxy, or N(R)₂, wherein each R is hydrogen, (C₁-C₄)alkyl, phenyl,COOH, CO₂(C₁-C₄)alkyl, or O[(C₁-C₄)alkyl], or a pharmaceuticallyacceptable salt thereof.

Another specific aspirinate useful in the present invention is acompound of formula II, III, IV or V wherein R¹ is hydrogen; or apharmaceutically acceptable salt thereof.

Another specific aspirinate useful in the present invention is acompound of formula II, III, IV or V wherein R¹ is nitro, halo, cyano,hydroxy, or N(R)₂, wherein each R is hydrogen, (C₁-C₄)alkyl, phenyl,COOH, CO₂(C₁-C₄)alkyl, or O[(C₁-C₄)alkyl]; or a pharmaceuticallyacceptable salt thereof.

Another specific aspirinate useful in the present invention is acompound of formula II, III, IV or V wherein X is S.

Another specific aspirinate useful in the present invention is acompound of formula II, III, IV or V wherein Y is S.

The compounds of formulas (I), like aspirin, can transfer acetylfunctionality. Moreover, the thiophene ring skeleton of compounds offormulas (I), which is similar in size to the benzene ring system inaspirin, results in a similar biodistribution, pharmacokinetics andpharmacodynamics for these compounds relative to aspirin. Furthermore,the thiophene ring sulfur (n=0) in a compound of formula I can bereadily catabolized to sulfone (n=2) and sulfoxide (n=1), whichincreases the water solubility of the compounds, so they can be rapidlyexcreted. This rapid catabolism reduces the gastric irritation, gastriculcers and occasional bleeding observed with high doses of aspirin, aswell as kidney retention leading to crystal urea and kidney stones, allof which are due to the insolubility of salicylates and divalent andtrivalent complexes of salicylates with metals. Besides being useful asTGF-beta elevating agents, the compounds of formula (I) are useful asanti-inflammatory agents, e.g., as anti-platelet aggregation agents,thrombin inhibitory agents, and vascular smooth muscle cellanti-proliferative agents.

Furthermore, substitution of electron withdrawing and electron donatingfunctionalities on the thiophene ring system can enhance or diminish thebioavailability of the substituted compounds. Thus, some of thesubstituted compounds exhibit higher protein binding affinities, andthus have higher binding affinities to serum proteins. The higherbinding affinities lead to a longer serum half-life, which provides alonger duration of action for the compounds. Other substituted compoundsexhibit lower protein binding affinities, and thus have lower bindingaffinities to serum proteins. The lower binding affinities lead to ashorter serum half-life, which provides a shorter duration of action forthe compounds. Moreover, the compounds of formula (I) can chelate metalions, which can result in enhanced transport across membranes.

The aspirinates of the invention preferably include copper salts, aswell as alkali metal or alkaline earth metal aspirinate salts, such aslithium, sodium, potassium, magnesium, zinc, or calcium aspirinatesalts, although other salts are envisioned.

The copper aspirinate salts of the invention can be formed for exampleby reacting a copper salt such as cupric chloride with the sodium saltsof 3,5-diisopropyl salicylic acid, acetylsalicylic acid, salicylic acid,3,5-ditertiary butyl salicylic acid, adamantylsalicylic acid,3,5-dibromoacetylsalicylic acid, 3,5-diiodoacetylsalicylic acid,4-tertiary butylsalicylic acid, 4-nitrosalicylic acid, 4-aminosalicylicacid, 4-acetylaminosalicylic acid, 5-chlorosalicylic acid and3,5-dichlorosalicylic acid.

The copper salt of a thiophene-ring based analog or derivative of anaspirinate of the invention can be prepared by reacting a copper salt,e.g., cupric chloride, with the sodium salt of the thiophene-basedanalog or derivative.

Inorganic copper salts useful in synthesizing copper aspirinate salts ofthe invention include hydrated copper chloride, and the dehydratethereof, hydrated copper fluoride and the dehydrate thereof, copperfluorosilicate and the hexahydrate thereof, copper sulfate and thepentahydrate thereof, copper nitrate and the tri- and hexa-hydratesthereof, copper bromide, copper metaborate, copper bromate, copperchlorate, copper iodate and copper fluorophosphate. In the above salts,the copper is typically in the Copper (II) oxidation state.

It is preferable to produce copper aspirinate coordination solvatesrather than anhydrous compounds. The copper aspirinate compounds may besolvated with a lower alkanol, e.g., a C₂-C₆ aliphatic alkanol such asethanol or isopropanol, a ketone such as acetone or methylethylketone,alkanolamines, pyridine, water, dimethyl formamide, or dimethylsulfoxide.

Compounds of Formula (VI) Falling Within the Scope of the Invention.

A specific compound of formula VI is a compound wherein — is a singlebond.

Another specific compound of formula VI is a compound wherein R⁹ andR^(j) together are —CH₂CH₂—, —S—, —O— —N(H)—, —N[(C₁-C₆)alkyl]—, or—CH═CH—.

Another specific compound of formula VI is a compound wherein — is—C(B)(D)—, wherein B and D are each halogen; and R⁸ and R⁹ are bothhydrogen.

Another specific compound of formula VI is a compound wherein R⁶ is notphenyl or phenyl substituted by 1 or 2 V. Another specific compound offormula VI is a compound wherein R⁷ is not phenyl or phenyl substitutedby 1 or 2 V. Another specific compound of formula VI is a compoundwherein R⁸ is not phenyl, or phenyl substituted by 1 or 2 V.

A specific value for Z is —(CH₂)₁₋₃—, —O—, —OCH₂—, —CH₂O—, —C(═O)O—,—N(R_(q))—, or a covalent bond. Another specific value for Z is —O—,—OCH₂—, —CH₂O—, —C(═O)O—, or —N(R_(q))—.

A specific compound of formula VI is a compound of formula VII:

wherein

Z is C═O or a covalent bond;

R¹⁰ is mercapto, (C₁-C₄)alkylthio, hydroxy, (C₁-C₆)alkoxy,—O(CH₂)₁₋₄COOH, —S(CH₂)₁₋₄COOH, —(CH₂)₀₋₄COOH, —O(CH₂)₂₋₄OH,—S(CH₂)₂₋₄OH, —O(CH₂)₁₋₄(C═O)R_(r), —S(CH₂)₁₋₄(C═O)R_(r),—O(CH₂)₂₋₄R_(r), —S(CH₂)₂₋₄R_(r), —(CH₂)₀₋₄R_(r), or—(CH₂)₀₋₄C(═O)R_(r);

R¹¹ is 3-(R_(s))-4-(R_(t))phenyl, halo(C₁-C₁₂)alkyl, (C₁-C₁₂)alkyl,(C₃-C₆)cycloalkyl, (C₁-C₆)alkylcyclo(C₁-C₆)alkyl, (C₃-C₆)cycloalkenyl,or (C₁-C₆)alkyl(C₃-C₆)cycloalkenyl;

R¹² is nitro, halo, ethyl,2-cyanoethyl, 2-trifluoromethylethyl,—CH₂CH₂C(═O)O(C₁-C₄)alkyl, chloroethyl, cyclohexane, or naphthlene;

R¹³ is H or together with R¹² is O—CH═CH—, —CH₂—CH₂— or —S—,

R¹⁴ is hydrogen, iodo, O(C₁-C₄)alkyl, hydroxy, —C(═O)O(C₁-C₆)alkyl,—OC(═O)(C₁-C₆)alkyl, benzyl, or OSO₂(CH₂)₀₋₄CH₃;

R¹⁵ is hydrogen, (C₁-C₆)alkyl, mercapto, (C₁-C₄)alkylthio, hydroxy,(C₁-C₆)alkoxy, iodo, OPO₃H₂, —OSO₂(CH₂)₀₋₄CH₃, —C(═O)O(C₁-C₆)alkyl,—OC(═O)(C₁-C₆)alkyl, or benzyl;

R_(r) is amino, optionally substituted with one or two (C₁-C₆)alkyl; orR_(r) is an N-heterocyclic ring which optionally comprises anotherhetero atom selected from N, O, or S in said ring;

R_(s) is hydrogen, halo, or hydroxy; and

R_(t) is hydrogen, (C₁-C₆)alkyl, mercapto, (C₁-C₄)alkylthio, hydroxy,(C₁-C₆)alkoxy, —OSO₂—(CH₂)₀₋₄—CH₃, halo, —OC(═O)(C₁-C₆)alkyl, or benzyl;

the compound is MER25, zindoxifene, DDAC (Analog II) or DTAC (102b);

a pharmaceutically acceptable salt thereof, or mixtures thereof.

A preferred compound of formula VII useful in the present invention is acompound wherein R¹⁴ is at the 5-position of the phenyl ring to which itis attached.

Another specific compound of formula (VI) useful in the presentinvention is a compound of formula (VII):

wherein

A is O or S;

Z is C═O or a covalent bond;

R¹⁶ and R¹⁷ are individually (C₁-C₄)alkyl or together with N are asaturated heterocyclic ring, preferably a 5-7 membered heterocyclic ringoptionally containing 1-2 additional N(R_(u)), S or nonperoxide O,wherein R_(u) is hydrogen, (C₁-C₄)alkyl, phenyl or benzyl;

R¹⁸ is hydrogen, (C₁-C₆)alkyl, mercapto, (C₁-C₄)alkylthio, hydroxy,(C₁-C₆)alkoxy;

R¹⁹ is nitro, halo, ethyl, 2-cyanoethyl, 2-trifluoromethylethyl,—CH₂CH₂C(═O)O(C₁-C₄)alkyl, or chloroethyl;

R²⁰ is H or together with R¹⁹ is —CH₂—CH₂— or —S—;

R²¹ is hydrogen, iodo, hydroxy, or O(C₁-C₄)alkyl;

R²² is hydrogen, (C₁-C₆)alkyl, mercapto, (C₁-C₄)alkylthio, hydroxy,(C₁-C₆)alkoxy, halo, or OPO₃H₂;

the compound is MER25, zindoxifene, DDAC (Analog II) or DTAC (102b);

a pharmaceutically acceptable salt thereof, or mixtures thereof.

Another specific compound of formula (VI) useful in the presentinvention is a compound of formula (VII):

wherein

A is O;

Z is C═O or a covalent bond;

R¹⁶ and R¹⁷ are individually (C₁-C₄)alkyl or together with N are asaturated heterocyclic ring, preferably a 5-7 membered heterocyclic ringoptionally containing 1-2 additional N(R_(n)), S or nonperoxide O,wherein R_(u) is hydrogen, (C₁-C₄)alkyl, phenyl or benzyl;

R¹⁸ is hydrogen, hydroxy, (C₁-C₄)alkyl, or (C₁-C₄)alkoxy;

R¹⁹ is nitro, halo, ethyl or chloroethyl;

R²⁰ is H or together with R¹⁹ is —CH₂—CH₂— or —S—;

R²¹ is hydrogen, iodo, hydroxy, or (C₁-C₄)alkoxy;

R²² is iodo, OPO₃H₂, (C₁-C₄)alkoxy or hydrogen;

the compound is MER25, zindoxifene, DDAC (Analog II) or DTAC (102b);

a pharmaceutically acceptable salt thereof, or mixtures thereof.

A preferred compound of formula VIII useful in the present invention isa compound wherein Z is a covalent bond; R¹⁶ and R¹⁷ are each(C₁-C₄)alkyl or —(CH₂)_(m)—; R¹⁸ is hydrogen; R²¹ is hydrogen or iodo;and m is 4-6.

A preferred compound of formula VIII useful in the present invention isa compound wherein R¹⁹ is ethyl or chloroethyl.

A preferred compound useful in the present invention is a compound offormula VIII wherein R¹⁹ and R²⁰ together are —CH₂—CH₂—; and R²² isOCH₃.

A preferred compound of formula VIII useful in the present invention isa compound wherein:

Z is C═O or a covalent bond;

R¹⁶ and R¹⁷ are individually (C₁-C₄)alkyl or together with N are asaturated heterocyclic ring, preferably a 5-7 membered heterocyclic ringoptionally comprising 1-2 additional N(R), S or nonperoxide O, wherein Ris hydrogen, (C₁-C₄)alkyl, phenyl or benzyl;

R¹⁸ is hydrogen, hydroxy or O(C₁-C₄)alkyl;

R¹⁹ is ethyl or chloroethyl;

R²⁰ is H or together with R¹⁹ is —CH₂—CH₂— or —S—;

R²¹ is hydrogen, iodo, hydroxy, or O(C₁-C₄)alkyl;

R²² is iodo, OPO₃H₂, O(C₁-C₄)alkyl or hydrogen;

a pharmaceutically acceptable salt thereof, or mixtures thereof.

Additionally for any compound of formula VIII or preferred compound offormula VIII described above, a specific value for R¹⁸ is hydrogen; forZ is a covalent bond; for R¹⁶ and R¹⁷ is independently (C₁-C₄)alkyl, orfor R¹⁶ and R¹⁷ taken together is —(CH₂)_(m)—; for R²¹ is hydrogen oriodo; and for m is 4-6.

Additionally for any compound of formula VIII or preferred compound offormula VIII described above, a specific value for R²² is OCH₃; and forR¹⁹ and R²⁰ together is —CH₂—CH₂—.

Compounds of formula VI useful in the present invention includetamoxifen and structural analogs of tamoxifen having substantialequivalent bioactivity. Such analogs include idoxifene, raloxifene,droloxifene, 3-iodotamoxifen, 4-iodotamoxifen, tomremifene, trioxifene,nafoxidene, 4-hydroxytamoxifen, H-1285, and pharmaceutically acceptablesalts thereof. A preferred embodiment of the invention is a compound offormula (VIII) wherein R¹⁹ is not ethyl when R²⁰, R²¹, and R²² are H.

The term “structural analogs thereof” with respect to tamoxifenincludes, but is not limited to, all of the compounds of formula (VI)which are capable of enhancing, increasing or elevating the level ofTGF-beta. See, for example, U.S. Pat. Nos. 4,536,516, 5,457,113,5,047,431, 5,441,986, 5,426,123, 5,384,332, 5,453,442, 5,492,922,5,462,937, 5,492,926, 5,254,594 and U.K. Patent 1,064,629.

Because tamoxifen (TMX) causes liver carcinogenicity in rats and hasbeen correlated with an increased risk of endometrial cancer in womenand may increase the risk of certain gut cancers, other tamoxifenanalogs may be considered safer to administer if they are lesscarcinogenic. The carcinogenicity of TMX has been attributed to theformation of covalent DNA adducts. Of the TMX analogs and derivatives,only TMX and toremifene have been studied for long-term carcinogenicityin rats. These studies provide strong evidence that covalent DNA adductsare involved in rodent hepatocarcinogenicity of TMX. Toremifene, whichexhibits only a very low level of hepatic DNA adducts, was found to benon-carcinogenic. See Potter et al., Carcinogenesis, 15, 439 (1994).

It is postulated that 4-hydroxylation of TMX yields electrophilicalkylating agents which alkylate DNA through the ethyl group of TMX.This mechanistic hypothesis explains the low level of DNA adductformation by the non-TMX analogs of formula (VI), including the TMXanalog toremifene, and the absence of DNA adducts detected for theanalogs 4-iodotamoxifen and idoxifene. Thus, all of these analogs arelikely to be free from the risk of significant carcinogenesis in longterm use. See Potter et al., supra. Idoxifene (IDX) includes(E)-1-[4-[2-(N-pyrrolidino)ethoxy]phenyl]-1-(4-iodophenyl)-2-phenyl-1-buteneand its pharmaceutically acceptable salts and derivatives. See R.McCague et al., Organic Preparations and Procedures Int., 26, 343 (1994)and S. K. Chandler et al., Cancer Res., 51, 5851 (1991). Besides itslower potential for inducing carcinogenesis via formation of DNA adductswhich can damage DNA, other advantages of IDX compared with TMX are thatIDX has reduced residual estrogenic activity in rats and an improvedmetabolic profile.

Other “antisteroids” or “steroidal antagonists” are useful as TGF-betaactivators or production stimulators or lead compounds, including otherknown stilbene-type antisteroids such as for example, cis- andtrans-clomiphene, toremifene, centchroman, raloxifene, droloxifene,(1-[4-(2-dimethylaminoethoxy)phenyl]-1-(3-hydroxyphenyl)-2-phenyl-2-butene(see U.S. Pat. No. 5,384,332), 1-nitro-1-phenyl-2-(4-hydroxyphenyl oranisyl)-2-[4-(2-pyrrol-N-ylethoxy)-phenyl]ethylene(CN-55,945),trans-1,2-dimethyl-1,2-(4-hydroxyphenyl)ethylene(trans-dimethylstilboestrol),trans-diethylstilboestrol, and1-nitro-1-phenyl-2-(4-hydroxyphenyl)-2-[4-(3-dimethylaminopropyloxy)phenyl-ethylene(GI680), metabolites or pharmaceutically acceptable salts thereof.

Known 1,2-diphenylethane-type antisteroids includecis-1,2-anisyl-1-[4-(2-diethylaminoethoxy)phenyl]ethane (MRL-37),1-(4-chlorophenyl) 1-[4-(2-diethylaminoethoxy)phenyl]-2-phenylethanol(WSM-4613);1-phenyl-1-1-[4-(2-diethylaminoethoxy)phenyl]-2-anisylethanol (MER-25);1-phenyl-1-[4-(2-diethylaminoethoxy)phenyl]-2-anisyl-ethane,mesobutoestrol (trans-1,2-dimethyl-1,2-(4-hydroxyphenyl)-ethane),meso-hexestrol, (+)hexestrol and (−)-hexestrol.

Known naphthalene-type antisteroids include nafoxidine,1-[4-(2,3-dihydroxypropoxy)phenyl]-2-phenyl-6-hydroxy-1,2,3,4-tetrahydro-naphthalene,1-(4-hydroxyphenyl)-2-phenyl-6-hydroxy-1,2,3,4-tetrahydronaphthalene,1-[4-(2-pyrrol-N-ylethoxy)-phenyl]-2-phenyl-6-methoxy-3,4-dihydronaphthalene(U11, 100A), and1-[4-(2,3-dihydroxypropoxy)phenyl]-2-phenyl-6-methoxy-3,4-dihydronaphthalene(U-23, 469).

Known antisteroids which do not fall anywhere within these structuralclassifications include coumestrol, biochanin-A, genistein,methallenstril, phenocyctin, and1-[4-(2-dimethylaminoethoxy)phenyl]-2-phenyl-5-methoxyindene (U, 11555).In the nomenclature employed hereinabove, the term “anisyl” is intendedto refer to a 4-methoxyphenyl group.

Preparation of a Compound of Formula (I-V)

Generally, a compound of formula I wherein R⁴ is (═O)_(n) and n is 0 maybe prepared by processes which are well known in the chemical arts forthe synthesis of thiophene compounds and other aromatic compounds.

A compound of formula I wherein R⁴ is (═O)_(n) and n is 1 or 2 can beprepared from a corresponding compound of formula II wherein n is 0, byoxidation of the thiophene sulfur using standard oxidation conditions.

Compounds of formula I wherein R⁴ is (C₁-C₆)alkyl, (C₁-C₆)alkanoyl or(C₂-C₆)alkanoyloxy and forms a sulfonium salt with the thiophene sulfur,wherein the associated counter ion is a pharmaceutically acceptableanion, can be prepared from corresponding compounds of formula I whereinR⁴ is (═O)_(n) and n is 0 by alkylation or acylation of the thiophenesulfur, using procedures which are well known in the art.

The synthesis of a compound of formula (II) can be carried out asfollows:

The synthesis of a compound of formula (III) may be carried out asfollows:

The synthesis of a compound of formula (IV) may be carried out asfollows:

The synthesis of a compound of formula (V) may be carried out asfollows:

Preparation of Compounds of Formula (VI)

Generally, compounds of formula (VI) may be prepared using synthetictechniques which are analogous to techniques known in the art, includingtechniques described in R. A. Magarian, Current Medicinal Chemistry,1994, 1, 61-104 and techniques described in the references relating totamoxifen analogs which are cited and incorporated herein.

It may be convenient to optionally use a conventional protecting groupduring the preparation of compounds of formula (I) or compounds offormula (VI). The protecting group may be removed at an appropriate timeduring the synthesis, such as for example, when the final compound is tobe formed. Such processes and intermediates for the manufacture of acompound of formula I are provided as further features of the invention.

Pharmaceutically Acceptable Acid and Base Addition Salts

The compounds used in the methods of the invention form pharmaceuticallyacceptable acid and base addition salts with a wide variety of organicand inorganic acids and bases and include the physiologically acceptablesalts which are often used in pharmaceutical chemistry. Such salts arealso part of this invention. Typical inorganic acids used to form suchsalts include hydrochloric, hydrobromic, hydroiodic, nitric, sulfuric,phosphoric, hypophosphoric and the like. Salts derived from organicacids, such as aliphatic mono and dicarboxylic acids, phenyl substitutedalkanoic acids, hydroxyalkanoic and hydroxyalandioic acids, aromaticacids, aliphatic and aromatic sulfonic acids, may also be used. Suchpharmaceutically acceptable salts thus include acetate, phenylacetate,trifluoroacetate, acrylate, ascorbate, benzoate, chlorobenzoate,dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate,o-acetoxybenzoate, naphthalene-2-benzoate, bromide, isobutyrate,phenylbutyrate, β-hydroxybutyrate, butyne-1,4-dioate, hexyne-1,4-dioate,caprate, caprylate, chloride, cinnamate, citrate, formate, fumarate,glycollate, heptanoate, hippurate, lactate, malate, maleate,hydroxymaleate, malonate, mandelate, mesylate, nicotinate,isonicotinate, nitrate, oxalate, phthalate, terphthalate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, propiolate, propionate, phenylpropionate, salicylate,sebacate, succinate, suberate, sulfate, bisulfate, pyrosulfate, sulfite,bisulfite, sulfonate, benzene-sulfonate, p-bromophenylsulfonate,chlorobenzenesulfonate, ethanesulfonate, 2-hydroxyethanesulfonate,methanesulfonate, naphthalene-1, sulfonate, naphthalene-2-sulfonate,p-toluenesulfonate, xylenesulfonate, tartarate, and the like. Apreferred salt is the hydrochloride salt.

The pharmaceutically acceptable acid addition salts are typically formedby reacting a compound of formula (I) or (VI) with an equimolar orexcess amount of acid. The reactants are generally combined in a mutualsolvent such as diethyl ether or benzene. The salt normally precipitatesout of solution within about one hour to 10 days and can be isolated byfiltration or the solvent can be stripped off by conventional means.

Bases commonly used for formation of acid salts include ammoniumhydroxide and alkali and alkaline earth metal hydroxide, carbonates, aswell as aliphatic and primary, secondary and tertiary amines, aliphaticdiamines. Bases especially useful in the preparation of addition saltsinclude ammonium hydroxide, potassium carbonate, methylamine,diethylamine, ethylene diamine, cyclohexylamine and ethanolamine.

The pharmaceutically acceptable salts generally have enhanced solubilitycharacteristics compared to the compound from which they are derived,and thus are often more amenable to formulation as liquids or emulsions,and can have enhanced bioavailability.

Identification of Therapeutic Agents Falling within the Scope of theInvention

Therapeutic agents useful in the practice of the invention, i.e., agentsthat elevate or increase TGF-beta levels, can be identified by an invitro assay described in copending U.S. application Ser. No. 08/476,735,the disclosure of which is incorporated by reference herein, and/or theassays described in the Examples hereinbelow. It is recognized that notall therapeutic agents, e.g., aspirinates, can increase TGF-beta levels.Moreover, it is recognized that some therapeutic agents within the scopeof the invention increase TGF-beta levels to a greater extent than otherTGF-beta elevating agents, however, methods to determine whether anagent falls within the scope of the invention are described hereinbelow.

The amounts of latent and/or active TGF-beta present in a sample ofphysiological fluid, such as a blood fraction, before and/or after theadministration of the therapeutic agent, can be measured by methodsdisclosed in copending U.S. application Ser. No. 08/477,393 and U.S.Pat. No. 5,545,569, issued Aug. 13, 1996, the disclosures of which areincorporated by reference herein.

For example, to determine whether an agent can elevate levels ofTGF-beta, an agent or mixture of agents is first tested on rat aorticvascular smooth muscle cells (rVSMCs) for their ability to stimulate theproduction of active TGF-β in the culture medium as originally describedfor tamoxifen. See Grainger et al. (Biochem. J., 294, 109 (1993)). Thekey step in demonstrating that cells have a reduced proliferation rateas a result of TGF-β production and activation is that the effect can befully reversed by neutralizing antibodies to TGF-β. Incomplete reversalof a decreased rate of proliferation is evidence for TGF-β independenteffect(s), which may include toxicity.

The effects of an agent are then tested on explant human aortic smoothmuscle cells (hVSMC) to determine whether the agent also stimulatesproduction of TGF-β by these cells. The use of explant hVSMCs, isessential because (I) explant hVSMCs grown under non-optimal conditions(particularly at low cell densities) will spontaneously produce TGF-β;(ii) hVSMC cultures from cells prepared by enzyme dispersalspontaneously produce substantial amounts of TGF-β in culture(Kirschenlohr et al., Am. J. Physiol., 265, C571 (1993)) and thereforecannot be used for screening; and (iii) the sensitivity of rVSMCs andhVSMCs to agents which induce the cells to produce TGF-β differs by upto 100-fold.

In screening for agents likely to be effective for clinical purposes, itis therefore necessary to use hVSMCs to determine both potency and thetherapeutic window between effective concentrations and toxicconcentrations for human cells. Candidate agents which pass the in vitrocell culture screens are then tested on one or more animal models ofvascular conditions or disease, e.g., animal models of atherosclerosisinclude lipid lesion formation in C57B16 mice and mice expressing thehuman apo(a) transgene that are fed a high fat diet, apoe knockout micefed a normal diet, or cholesterol-fed Watanabe rabbits.

To determine total TGF-beta, ELISA plates are coated with a chickenantibody that binds both latent and active TGF-beta. Patient sera orplasma are incubated with these ELISA plates, then the plates are washedto remove unbound components of the patients' sera or plasma. Rabbitanti-TGF-beta antibody, capable of binding both latent and activeTGF-beta, is then added to the plates and incubated. The plates are thenwashed to remove unbound antibody, and peroxidase-labeled anti-rabbitIgG is added. After incubation and washing, the plates are exposed tothe chromogenic substrate, ortho-phenylenediamine. The presence of totalTGF-beta in patients' sera or plasma is then determined calorimetricallyat A₄₉₂ by comparison to a standard curve. In patients treated with anagent that modifies TGF-beta, a pretreatment determination of TGF-betacan be compared with post-treatment time points to monitor treatmentresults and effectiveness.

In an alternate format, TGF-beta type II receptor extracellular domain,which recognizes the active form(s) of TGF-beta, but not the mature orlatent forms, is coated onto ELISA plates. Patient sera or plasma areadded to the plates, and processed as above. This assay measures activeTGF-beta present in sera or plasma.

In another alternate format, fluorescent-labeled anti-TGF-beta antibodyis used in place of peroxidase labeled second antibody to detect thepresence of TGF-beta in patients' sera or plasma. In yet anotheralternate format, anti-TGF-beta antibody is labeled with a radioactivemoiety capable of detection by standard means. These latter two assaysmay be performed in an ELISA format, with or without using theadditional anti-TGF-beta antibody described above.

It is envisioned that the therapeutic agents of the invention canincrease TGF-beta levels by increasing the number of TGF-betatranscripts, increasing the translational efficiency of TGF-betatranscripts, increasing the post-translational processing of the latentform of TGF-beta to the active form of TGF-beta, increasing thebioavailability of TGF-beta, and/or increasing the biological effect ofactive TGF-beta, e.g., by increasing the affinity of TGF-beta for itsreceptor, increasing the affinity of the receptor for TGF-beta and/or byincreasing the number of receptors for TGF-beta on the cell surface, orany combination thereof. For example, the administration of aspirin orcopper aspirinate (see Examples m and IV) can increase the level oflatent TGF-beta in a mammal relative to the level of latent TGF-beta inthat mammal prior to aspirin or copper aspirinate administration.

Agents useful in the practice of the methods of the invention can alsobe identified by the correlation of agent administration with theinhibition or reduction in atherosclerotic plaque development orformation, an increase in lesion regression or plaque stability, or adecrease vascular wall hypertrophy and/or hyperplasia in vivo. Agentefficacy is measured by methods available to those skilled in the artincluding, but not limited to, angiography, ultrasonic evaluation,fluoroscopic imaging, fiber optic endoscopic examination or biopsy andhistology. The activity of the therapeutic agents of the invention invivo can also be monitored indirectly by the measurement of the levelsof TGF-beta in a patient before and after the administration of thetherapeutic agent.

For non-vascular indications, agents useful in the practice of theinvention can be identified by the correlation of in vivo agentadministration with a reduction in a particular pathology associatedwith the non-vascular indication. For example, animal models formultiple sclerosis (Martin et al., Ann. Rev. Immunol., 10, 153 (1992);Hafler et al., Immunol. Today 10, 107 (1989), WO 93/16724) andrheumatoid arthritis (WO 93/16724) may be employed to determine theactivity of the therapeutic agents of the invention in vivo.Additionally, suitable animal models for osteoporosis (suspensioninduced osteoporosis in rats) and cancer (DMBA-induced skin cancer) arewell known in the art.

Vascular indications Amenable to Treatment by the claimed Methods

The therapeutic agents of the invention are useful to treat a mammalsuch as a human patient, afflicted with, or at risk of, a vascularindication. In particular, the therapeutic agents of the invention areuseful to treat a mammal afflicted with, or at risk of, a vascularindication associated with a deficiency in TGF-beta.

A mammal afflicted with, or at risk of, a vascular indication that wouldbenefit from the practice of the claimed invention includes a mammalexhibiting a reduced level of TGF-beta within the vessel wall. Suchmammals may be identified as having one or more risk factors whichcontribute to reduced TGF-beta activity. These factors include low serumactive levels of TGF-beta, elevated circulating PAI-1 antigen oractivity, elevated circulating lipoprotein (a), elevated bloodconcentration of LDL and/or VLDL in the fasting state, the ability toelevate PAI-1 following a fat tolerance test, the presence of the 4Gallele of the PAI-1 promoter, and the like. Thus, the measurement ofPAI-1/TGF-beta response (Example 7) to fat feeding is one method todetermine whether an individual is at risk of a vascular indicationassociated with a deficiency in TGF-beta levels. For example, low serumactive TGF-beta levels can be levels that are less than about 4 ng/ml,preferably less than about 3 ng/ml, and more preferably less than about2 ng/ml.

Dosages, Formulations and Routes of Administration of the TherapeuticAgents of the Invention

Aspirin or aspirinates, e.g., the compounds of formulas (I), and theaspirinate salts of the invention, including their coordinationsolvates, are preferably administered at doses of about 0.001-600 mg/kg,more preferably at doses of about 2.0-165 mg/kg, and even morepreferably at doses of about 1.0-100 mg/kg of body weight, althoughother dosages may provide beneficial results.

Fish oil, a source of omega-3 fatty acids, is administered at doses ofabout 200-18000 mg/kg/day, more preferably at doses of about 1000-6000mg/kg/day, and even more preferably at doses of about 1200-4000mg/kg/day, although other dosages may provide beneficial.

For compounds of the formula (VI), generally, accepted and effectivedaily doses will be from about 0.05 mg/kg/day to about 10 mg/kg/day,preferably about 0.1-1.0 mg/kg/day, more preferably about 0.3-0.5mg/kg/day. For local delivery, an exemplary dose will be about 0.01 toabout 1000 μg/ml, preferably followed by a chronic lower dose, which ispreferably administered orally. It is also contemplated that a largeloading dose may be employed, e.g., about 10 to about 100 mg/kg, torapidly establish a therapeutic level of the agent. The large loadingdose is preferably followed by a chronic dose of about 0.1 to about 20mg/kg/day, preferably about 0.5 to about 2 mg/kg/day. It is preferredthat a compound of formula (VI) is administered in the form of an acidaddition salt, as is customary in the administration of pharmaceuticalscomprising a basic group, such as an amino or N-heterocyclic group.

The amount of therapeutic agent administered is selected to treat aparticular vascular indication. For example, to treat vascular traumasof differing severity, smaller doses are sufficient to treat lesservascular trauma, such as to prevent vascular rejection following graftor transplant, while larger doses are sufficient to treat more extensivevascular trauma, such as restenosis following angioplasty. Thetherapeutic agents of the invention are also amenable to chronic use forprophylactic purposes to treat disease states involving proliferation ofvascular smooth muscle cells and pericytes derived from the mediallayers of vessels, pericytes and fibroblasts in the adventitia, andmigrating macrophage/monocyte/foam cells, over time (e.g.,atherosclerosis, coronary heart disease, thrombosis, myocardialinfarction, stroke, uterine fibroid or fibroma and the like), preferablyby systemic administration.

Administration of the therapeutic agents in accordance with the presentinvention may be continuous or intermittent, depending, for example,upon the recipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of the agents of theinvention may be essentially continuous over a preselected period oftime or may be in a series of spaced doses, e.g., either before, during,or after procedural vascular trauma, before and during, before andafter, during and after, or before, during and after the proceduraltrauma.

One or more suitable unit dosage forms comprising the therapeutic agentsof the invention, which, as discussed below, may be formulated forsustained release, can be administered by a variety of routes includingoral, or parenteral, including by rectal, transdermal, subcutaneous,intravenous, intramuscular, intrapulmonary and intranasal routes. Whenthe therapeutic agents of the invention are prepared for oraladministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. The total active ingredients in suchformulations comprise from 0.1 to 99.9% by weight of the formulation. By“pharmaceutically acceptable” it is meant the carrier, diluent,excipient, and/or salt must be compatible with the other ingredients ofthe formulation, and not deleterious to the recipient thereof.

Pharmaceutical formulations containing the therapeutic agents of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. For example, a copperaspirinate including copper 2-acetylsalicylate, or a compound of formula(I), as well as a compound of formula (VI), can be formulated withcommon excipients, diluents, or carriers, and formed into tablets,capsules, suspensions, powders, and the like. Examples of excipients,diluents, and carriers that are suitable for such formulations includethe following fillers and extenders such as starch, sugars, mannitol,and silicic derivatives; binding agents such as carboxymethyl cellulose,HPMC, and other cellulose derivatives, alginates, gelatin, andpolyinyl-pyrrolidone; moisturizing agents such as glycerol;disintegrating agents such as calcium carbonate and sodium bicarbonate;agents for retarding dissolution such as paraffin; resorptionaccelerators such as quaternary ammomum compounds; surface active agentssuch as cetyl alcohol, glycerol monostearate; adsorptive carriers suchas kaolin and bentonite; and lubricants such as talc, calcium andmagnesium stearate, and solid polyethyl glycols.

For example, tablets or caplets containing aspirinates of the inventioncan include buffering agents such as calcium carbonate, magnesium oxideand magnesium carbonate. Caplets and tablets can also include inactiveingredients such as cellulose, pregelatinized starch, silicon dioxide,hydroxypropyl methylcellulose, magnesium stearate, microcrystallinecellulose, starch, talc, titanium dioxide, benzoic acid, citric acid,corn starch, mineral oil, polypropylene glycol, sodium phosphate, andzinc stearate, and the like. Hard or soft gelatin capsules containingaspirinates of the invention can contain inactive ingredients such asgelatin, microcrystalline cellulose, sodium lauryl sulfate, starch,talc, and titanium dioxide, and the like, as well as liquid vehiclessuch as polyethylene glycols (PEGs) and vegetable oil. Moreover, theenteric coated caplets or tablets of the copper aspirinates of theinvention are designed to resist disintegration in the stomach anddissolve in the more neutral to alkaline environment of the duodenum.

The pharmaceutical formulations of the therapeutic agents of theinvention can take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension. Thetherapeutic agents of the invention can also be formulated as elixirs orsolutions for convenient oral administration or as solutions appropriatefor parenteral administration, for instance by intramuscular,subcutaneous or intravenous routes.

These formulations can contain pharmaceutically acceptable vehicles andadjuvants which are well known in the prior art. It is possible, forexample, to prepare solutions using one or more organic solvent(s) thatis/are acceptable from the physiological standpoint, chosen, in additionto water, from solvents such as acetone, ethanol, isopropyl alcohol,glycol ethers such as the products sold under the name “Dowanol”,polyglycols and polyethylene glycols, C₁-C₄ alkyl esters of short-chainacids, preferably ethyl or isopropyl lactate, fatty acid triglyceridessuch as the products marketed under the name “Miglyol”, isopropylmyristate, animal, mineral and vegetable oils and polysiloxanes.

The compositions according to the invention can also contain thickeningagents such as cellulose and/or cellulose derivatives. They can alsocontain gums such as xanthan, alginates, guar, or carbo gum or gumarabic, or alternatively thickeners such as polyethylene glycols,bentones and montmorillonites, and the like.

It is possible to add, if necessary, an adjuvant chosen fromantioxidants, surfactants, preservatives, film-forming, keratolytic orcomedolytic agents, perfumes and colorings. Also, other activeingredients may be added, whether for the conditions described or someother condition.

For example, among antioxidants, t-butylhydroquinone, butylatedhydroxyanisole, butylated hydroxytoluene and a-tocopherol and itsderivatives may be mentioned. The galenical forms chiefly conditionedfor topical application take the form of creams, milks, gels, dispersionor microemulsions, lotions thickened to a greater or lesser extent,impregnated pads, ointments or sticks, or alternatively the form ofaerosol formulations in spray or foam form or alternatively in the formof a cake of soap.

Additionally, the agents are well suited to formulation as sustainedrelease dosage forms and the like. The formulations can be soconstituted that they release the active ingredient only or preferablyin a particular part of the intestinal tract, possibly over a period oftime. The coatings, envelopes, and protective matrices may be made, forexample, from polymeric substances or waxes.

The therapeutic agents of the invention can be delivered via patches fortransdermal administration. See U.S. Pat. No. 5,560,922 for examples ofpatches suitable for transdermal delivery of a therapeutic agent.Patches for transdermal delivery can comprise a backing layer and apolymer matrix which has dispersed or dissolved therein a therapeuticagent effective for reducing vessel lumen diameter diminution, alongwith one or more skin permeation enhancers. The backing layer can bemade of any suitable material which is impermeable to the therapeuticagent. The backing layer serves as a protective cover for the matrixlayer and provides also a support function. The backing can be formed sothat it is essentially the same size layer as the polymer matrix or itcan be of larger dimension so that it can extend beyond the side of thepolymer matrix or overlay the side or sides of the polymer matrix andthen can extend outwardly in a manner that the surface of the extensionof the backing layer can be the base for an adhesive means.Alternatively, the polymer matrix can contain, or be formulated of, anadhesive polymer, such as polyacrylate or acrylate/vinyl acetatecopolymer. For long-term applications it might be desirable to usemicroporous and/or breathable backing laminates, so hydration ormaceration of the skin can be minimized.

Examples of materials suitable for making the backing layer are films ofhigh and low density polyethylene, polypropylene, polyurethane,polyinylchloride, polyesters such as poly(ethylene phthalate), metalfoils, metal foil laminates of such suitable polymer films, and thelike. Preferably, the materials used for the backing layer are laminatesof such polymer films with a metal foil such as aluminum foil. In suchlaminates, a polymer film of the laminate will usually be in contactwith the adhesive polymer matrix.

The backing layer can be any appropriate thickness which will providethe desired protective and support functions. A suitable thickness willbe from about 10 to about 200 microns.

Generally, those polymers used to form the biologically acceptableadhesive polymer layer are those capable of forming shaped bodies, thinwalls or coatings through which therapeutic agents can pass at acontrolled rate. Suitable polymers are biologically and pharmaceuticallycompatible, nonallergenic and insoluble in and compatible with bodyfluids or tissues with which the device is contacted. The use of solublepolymers is to be avoided since dissolution or erosion of the matrix byskin moisture would affect the release rate of the therapeutic agents aswell as the capability of the dosage unit to remain in place forconvenience of removal.

Exemplary materials for fabricating the adhesive polymer layer includepolyethylene, polypropylene, polyurethane, ethylene/propylenecopolymers, ethylene/ethylacrylate copolymers, ethylene/vinyl acetatecopolymers, silicone elastomers, especially the medical-gradepolydimethylsiloxanes, neoprene rubber, polyisobutylene, polyacrylates,chlorinated polyethylene, polyinyl chloride, vinyl chloride-vinylacetate copolymer, crosslinked polymethacrylate polymers (hydrogel),polyinylidene chloride, poly(ethylene terephthalate), butyl rubber,epichlorohydrin rubbers, ethylenvinyl alcohol copolymers,ethylene-vinyloxyethanol copolymers; silicone copolymers, for example,polysiloxane-polycarbonate copolymers, polysiloxanepolyethylene oxidecopolymers, polysiloxane-polymethacrylate copolymers,polysiloxane-alkylene copolymers (e.g., polysiloxane-ethylenecopolymers), polysiloxane-alkylenesilane copolymers (e.g.,polysiloxane-ethylenesilane copolymers), and the like; cellulosepolymers, for example methyl or ethyl cellulose, hydroxypropyl methylcellulose, and cellulose esters; polycarbonates;polytetrafluoroethylene; and the like.

Preferably, a biologically acceptable adhesive polymer matrix should beselected from polymers with glass transition temperatures below roomtemperature. The polymer may, but need not necessarily, have a degree ofcrystallinity at room temperature. Cross-linking monomeric units orsites can be incorporated into such polymers. For example, cross-linkingmonomers can be incorporated into polyacrylate polymers, which providesites for cross-linking the matrix after dispersing the therapeuticagent into the polymer. Known cross-linking monomers for polyacrylatepolymers include polymethacrylic esters of polyols such as butylenediacrylate and dimethacrylate, trimethylol propane trimethacrylate andthe like. Other monomers which provide such sites include allylacrylate, allyl methacrylate, diallyl maleate and the like.

Preferably, a plasticizer and/or humectant is dispersed within theadhesive polymer matrix. Water-soluble polyols are generally suitablefor this purpose. Incorporation of a humectant in the formulation allowsthe dosage unit to absorb moisture on the surface of skin which in turnhelps to reduce skin irritation and to prevent the adhesive polymerlayer of the delivery system from failing.

Therapeutic agents released from a transdermal delivery system must becapable of penetrating each layer of skin. In order to increase the rateof permeation of a therapeutic agent, a transdermal drug delivery systemmust be able in particular to increase the permeability of the outermostlayer of skin, the stratum corneum, which provides the most resistanceto the penetration of molecules. The fabrication of patches fortransdermal delivery of therapeutic agents is well known to the art.

The local delivery of the therapeutic agents of the invention can alsobe by a variety of techniques which administer the agent at or near thediseased or traumatized vascular site. Examples of site-specific ortargeted local delivery techniques are not intended to be limiting butto be illustrative of the techniques available. Examples include localdelivery catheters, such as an infusion or indwelling catheter, a needlecatheter, shunts and stents or other implantable devices, site specificcarriers, direct injection, or direct applications. In addition, localdelivery of the therapeutic agents to branch points may be particularlybeneficial as active TGF beta levels are lower at branch points, wherelesion formation is increased relative to non-branch points.

Catheters which may be useful in the practice of the invention includecatheters such as those disclosed in Just et al. (U.S. Pat. No.5,232,444), Abusio et al. (U.S. Pat. No. 5,213,576), Shapland et al.(U.S. Pat. No. 5,282,785), Racchini et al. (U.S. Pat. No. 5,458,568) andShaffer et al. (U.S. Pat. No. 5,049,132), the disclosures of which areincorporated by reference herein.

For a compound of formula (VI), which may be administered in accordancewith the present invention using an infusion catheter, such as producedby C.R. Bard Inc., Billerica, Mass., or that disclosed by Wolinsky (U.S.Pat. No. 4,824,436) or Spears (U.S. Pat. No. 4,512,762), atherapeutically/-prophylactically effective dosage of the compounds offormula (VI) will be typically reached when the concentration thereof inthe fluid space between the balloons of the catheter is in the range ofabout 10⁻³ to 10⁻¹²M. The compounds of formula (VI) may only need to bedelivered in an anti-proliferative therapeutic/prophylactic dosagesufficient to expose the proximal (6 to 9) cell layers of the intimal ortunica media cells lining the lumen thereto. Also, such a dosage can bedetermined empirically, e.g., by a) infusing vessels from suitableanimal model systems and using immunohistochemical methods to detect thecompound of formula (VI) and its effects; and b) conducting suitable invitro studies.

Local delivery by an implant involves the surgical placement of a matrixthat contains the therapeutic agent at the lesion site or traumatizedarea. The implanted matrix releases the therapeutic agent by diffusion,chemical reaction, or solvent activators. Lange, Science, 249, 1527(1990).

An example of targeted local delivery by an implant is the use of astent. Stents are designed to mechanically prevent the collapse andreocclusion of the coronary arteries. Incorporating a therapeutic agentinto the stent delivers the therapeutic agent directly to the lesion.Local delivery of agents by this technique is described in Koh,Pharmaceutical Technology (October, 1990).

For example, a metallic, plastic or biodegradable intravascular stentcan be employed which comprises an effective amount of a therapeuticagent. The stent preferably comprises a biodegradable coating or aporous or permeable non-biodegradable coating comprising the therapeuticagent. A more preferred embodiment of the invention is a coated stentwherein the coating comprises a sustained-release dosage form of thetherapeutic agent. In an alternative embodiment, a biodegradable stentmay also have the therapeutic agent impregnated therein, i.e., in thestent matrix.

A biodegradable stent with the therapeutic agent impregnated therein canfurther be coated with a biodegradable coating or with a porousnon-biodegradable coating having the sustained release-dosage form ofthe therapeutic agent dispersed therein. Such a stent can provide adifferential release rate of the therapeutic agent, i.e., there can be afaster initial release of the therapeutic agent from the coatingfollowed by a slower delayed release of the therapeutic agentimpregnated in the stent matrix, upon degradation of the stent matrix.The intravascular stent also provides a mechanical means of providing anincrease in luminal area of a vessel.

Furthermore, the placement of intravascular stents comprising atherapeutic agent which is an inhibitor of smooth muscle cellproliferation can provide increased efficacy by reducing or preventingintimal proliferation. This inhibition of intimal smooth muscle cellsand stroma produced by the smooth muscle and pericytes can allow morerapid and complete re-endothelization following the intraventionalplacement of the vascular stent. The increased rate ofre-endothelization and stabilization of the vessel wall following stentplacement can reduce the loss of luminal area and decreased blood flowwhich is the primary cause of vascular stent failures.

Another example is a delivery system in which a polymer that containsthe therapeutic agent is injected into the lesion in liquid form. Thepolymer then solidifies or cures to form the implant which is retainedin situ. This technique is described in published PCT application WO90/03768 (Donn, Apr. 19, 1990).

Another example is the delivery of a therapeutic agent by polymericendoluminal sealing. This technique uses a catheter to apply a polymericimplant to the interior surface of the lumen. The therapeutic agentincorporated into the biodegradable polymer implant is thereby releasedat the surgical site. This technique is described in published PCTapplication WO 90/01969 (Schindler, Aug. 23, 1989), the disclosure ofwhich is incorporated by reference herein.

Yet another example of local delivery by an implant is by directinjection of vesicles or microparticulates into the lesion. Thesemicroparticulates may be composed of substances such as proteins,lipids, carbohydrates or synthetic polymers. These microparticulateshave the therapeutic agent incorporated throughout the microparticle orover the microparticle as a coating. Delivery systems incorporatingmicroparticulates are described in Lange, Science, 249,1527 (1990) andMathiowitz et al., J. App. Poly. Sci., 26, 809 (1981).

Local delivery by site specific carriers involves attaching thetherapeutic agent to a carrier which will direct the therapeutic agentto the target site, i.e., to a proliferative lesion. Examples of thisdelivery technique includes the use of carriers such as a proteinligand, e.g., a monoclonal antibody or antibody fragment. Lange,Science, 249,1527 (1990).

Local delivery by direct application also includes applying thetherapeutic agent directly to tissue, such as to the arterial bypassgraft during the surgical procedure, or an artificial graft, and thenimplanting the treated graft or other tissue.

For topical administration, the therapeutic agents may be formulated asis known in the art for direct application to a target area.Conventional forms for this purpose include wound dressings, coatedbandages or other polymer coverings, ointments, lotions, pastes,jellies, sprays, and aerosols. The percent by weight of a therapeuticagent of the invention present in a topical formulation will depend onvarious factors, but generally will be from 0.5% to 95% of the totalweight of the formulation, and typically 1-25% by weight.

It will be recognized by those skilled in the art thattherapeutically/-prophylactically effective dosages of these therapeuticagents and compositions will be dependent on several factors. Forexample, with respect to catheter delivery, those factors include a) theatmospheric pressure applied during infusion; b) the time over which theagent administered resides at the vascular site; c) the form of thetherapeutic or prophylactic agent employed; and/or d) the nature of thevascular trauma and therapy desired. Those skilled practitioners trainedto deliver drugs at therapeutically or prophylactically effectivedosages (e.g., by monitoring drug levels and observing clinical effectsin patients) will determine the optimal dosage for an individual patientbased on experience and professional judgment. Those skilled in the artwill recognize that infiltration of the therapeutic agent into intimallayers of a diseased or traumatized human vessel wall in free orsustained-release form is subject to variation and will need to bedetermined on an individual basis.

The invention will be better understood by making reference to thefollowing specific examples.

EXAMPLE I Association of TGF-beta with Lipoprotein Particles

TGF-beta is a hydrophobic protein known to have affinity for polymericaliphatic hydrocarbons. To determine whether TGF-beta would associatewith lipoprotein particles in the circulation, platelet-poor plasma wasprepared from peripheral venous blood drawn from ten healthy donors(A-J) and two donors with diabetes (K and L). The absence of plateletdegranulation (<0.02% degranulation) was confirmed by measurement ofPF-4 (platelet factor-4) in the plasma by ELISA (Asserchrom PF-4;Diagnostic Stago, FR). A 1 ml aliquot of plasma was diluted to 4 ml withBuffer A (Havel et al., J. Clin. Investig., 34, 1345 (1955)) and thenKBr was added to final density of 1.215 g/ml. The lipoproteins wereseparated from the plasma proteins by density gradientultracentrifugation (235,000× g) at 4° C. for 48 hours. The top 2 ml wascollected as the lipoprotein fraction and the lower 2 ml was collectedas the lipoprotein deficient plasma fraction.

The total cholesterol in each fraction was measured by the cholesteroloxidase enzymatic method (Sigma Diagnostics) as previously described inGrainger et al., Nat. Med., 1, 1067 (1995). The cholesterol in fractions0-9 was assumed to be VLDL, in fractions 10-19 to be LDL, and infractions 20-30 to be HDL, in accordance with the elution positions ofthe major apolipoproteins. Lipoprotein concentrations are reported as mMcholesterol. For cell cultures studies, the lipoprotein fraction wassubjected to extensive dialysis against serum-free DMEM, and the amountof TGF-beta was measured in the lipoprotein fraction and in the plasmaprotein fractions after treatment with acid/urea, using the QuantikineELISA (R&D Systems) in accordance with the manufacturer's instructions.

In some individuals (7/10), TGF-beta was detected in the lipoproteinfraction as well as the lipoprotein deficient plasma fraction. Theproportion of the TGF-beta associated with lipoprotein varied from <1%to 39% with a mean of 16%. Thus, plasma TGF-beta, unlike most otherplasma proteins, can associate with lipoprotein particles.

TABLE 1 Age % associated LDL Individual (yrs) Sex TGF-beta VLDL (mM) HDLA 44 M 27 0.9 3.1 0.8 B 28 M <1 0.5 2.8 1.1 C 41 F 24 1.1 4.7 0.7 D 31 M<1 0.6 3.4 0.8 E 28 M 7 0.3 3.0 0.9 F 21 F 19 1.1 2.6 1.0 G 22 M 11 0.83.6 0.9 H 49 M 39 1.5 3.3 1.0 I 47 M <1 0.8 3.7 0.8 J 29 M 9 0.9 3.1 1.0K 36 M 78 4.6 3.1 0.9 L 27 M 96 1.1 3.8 1.1

To determine whether the TGF-beta associated with lipoprotein particleswas able to bind to the type II TGF-beta signaling receptor and exertbiological activity in vitro, the binding of recombinant TGF-beta to R2Xwas measured in the absence and presence of increasing concentrations oflipoprotein purified from the plasma of an individual with <1 ng/mlTGF-beta in plasma (individual I, Table 1). If thelipoprotein-associated fraction of TGF-beta is unavailable for binding,lipoproteins prepared from an individual with a very low plasmaconcentration of TGF-beta would be expected to reduce the binding ofrecombinant active TGF-beta to its receptors. The half maximal (ka)binding of recombinant TGF-beta to the recombinant extracellular domainof the type II TGF-beta receptor was previously determined to be 17±3ng/ml (R2X; Grainger et al., Nature, 270, 460 (1994); Grainger et al.,Clin. Chim. Acta, 235, 11 (1995)).

To measure the binding of TGF-beta to its receptor, the recombinantextracellular domain of the type II TGF-beta receptor (R2X), was coatedonto ELISA plates (1 μg/well, Maxisorp plates, Gibco BRL). Wells werewashed 3 times quickly in TBS and blocked with TBS containing 3% bovineserum albumin (BSA, fatty-acid free; Sigma) for 30 minutes. A standardcurve of recombinant active TGF-beta1 (1.5 ng/ml to 100 ng/mlrecombinant active TGF-beta1 in two fold serial dilutions; R&D Systems)was prepared in TBS +0.1% BSA and in TBS +0.1% BSA additionallycontaining dialyzed lipoprotein at various concentrations. The standardcurves were incubated in the wells containing R2X for 2 hours. Theamount of bound TGF-beta was detected with antibody BDA5 (R&D Systems)as previously described by Grainger et al., Clin. Chim. Acta, 235, 11(1995). Briefly, after three quick washes with TBS, the wells wereincubated with TGF-beta detection antibody at 1 μg/ml in TBS +3% BSA (50μI/well) for 1 hour. After a further three washes in TBS, the wells wereincubated with anti-rabbit IgG conjugated to horseradish peroxidase(A-6154; Sigma) at 1:5000 dilution in TBS +3% BSA for 30 minutes. Thewells were washed 3 times with TBS and visualized using K-Blue Substrate(Elisa Technologies) for 20 minutes. All incubations were performed atroom temperature with shaking (˜300 rpm).

The presence of lipoprotein caused a dose-dependent increase in theapparent ka for TGF-beta binding to R2X to a maximal value of 42±6 ng/mlwhen lipoprotein equivalent to 3 mM total cholesterol was added (FIG.2A; values are the mean±standard error of triplicate determinations).The concentration of lipoprotein (measured as total cholesterol) whichhalf-maximally increased the apparent ka was approximately 1 mM. Thus,TGF-beta which is associated with lipoprotein particles has a loweraffinity for the type II TGF-beta receptor, or, if the TGF-beta is inequilibrium between the lipoprotein and aqueous phases, is unable tobind to the TGF-beta receptors.

It has previously been shown that TGF-beta inhibits the proliferation ofmink lung epithelial (MvLu) cells in culture. Recombinant activeTGF-beta1 was added to MvLu cells (passage 59-63 from the ATCC) whichwere growing in DMEM+10% fetal calf serum) and the concentration ofrecombinant TGF-beta required to half-maximally inhibit MvLu cells(reported as MvLu cell ID50) was measured as previously described(Danielpour et al., J. Cell Physiol., 138, 79 (1989); Kirschenlohr etal., Am. J. Physiol., 265, C571 (1993). Proliferation of MvLu cells washalf-maximally inhibited by recombinant active TGF-beta1 with an ID₅₀ of0.12±0.04 ng/ml (n=6) (FIG. 2B). Addition of lipoprotein purified fromthe plasma of individual I caused a dose-dependent increase in the ID₅₀of TGF-beta. The ID₅₀ was maximal at 0.52±0.08 ng/ml when 3 mM totalcholesterol was added. The concentration of lipoprotein whichhalf-maximally increased the ID₅₀ was approximately 0.8 mM. Therefore,TGF-beta associated with lipoprotein was less active, or inactive, as aninhibitor of MvLu cell proliferation.

Since low levels of TGF-beta activity have been associated with advancedatherosclerosis, individuals with a large proportion of their plasmaTGF-beta sequestered into an inactive lipoprotein-associated pool may beat significantly higher risk of developing the disease. The differencesin the proportion of TGF-beta associated with lipoprotein among theindividuals studied was therefore investigated further. The differentclasses of lipoprotein were separated by size using gel filtrationchromatography for ten healthy individuals A-J (Table 1) as well as twodiabetic individuals with abnormal lipoprotein profiles (individualsK-L, Table 1). The TGF-beta present in the fractions following the gelfiltration of the lipoprotein fraction from each of the ten individualswas then determined.

Individual A had a profile of lipoproteins typical of healthy subjects(FIG. 3A) and 27% of the plasma TGF-beta was associated with thelipoprotein fraction. 88% of the lipoprotein-associated TGF-beta elutedwith a tightly defined subfraction of the HDL particles, with thesmallest size of all the cholesterol-containing lipoprotein particles.The remaining 12% of the lipoprotein-associated TGF-beta was distributedamong the VLDL and LDL fractions. This pattern of association ofTGF-beta with a subfraction of HDL particles was typical of all thehealth donors tested (>80% of the lipoprotein-associated TGF-beta in asubfraction of HDL), except individual C.

Individual C had little VLDL or chylomicrons but moderately elevated LDLand 24% of the plasma TGF-beta was associated with the lipoprotein pool(FIG. 3B). As with the other individuals the majority (65%) of theTGF-beta was associated with the HDL subfraction. However, thisindividual had a significant amount of TGF-beta (27%) associated withLDL and the remainder eluted with the VLDL.

Individual K was a diabetic patient with hypertriglyceridaemia, and >50%of the total plasma cholesterol was present in the largesttriglyceride-rich lipoprotein particles (FIG. 3C). This individual had78% of the plasma TGF-beta associated with the lipoprotein pool, butonly 20% of this was present in the HDL subfraction. The remaining 80%co-eluted from the gel filtration column with the VLDL and chylomicrons.

Individual L was a diabetic patient with moderately elevated plasmatriglyceride and VLDL/chylomicrons and 92% of the plasma TGF-betaassociated with the lipoprotein (FIG. 3D). This individual had verylittle (<5%) of the lipoprotein-associated TGF-beta co-eluting with theHDL particles. Approximately 60% of the TGF-beta co-eluted with thelargest triglyceride-rich lipoprotein particles and the remainder withthe LDL particles.

Thus, TGF-beta associates with a subfraction of HDL particles which varyvery little in size and which are among the smallestcholesterol-containing lipoproteins present in plasma. Additionally,TGF-beta can associate with both the triglyceride-rich LDL and VLDLparticles (FIG. 10). Indeed, under conditions where the concentrationsof these particles in plasma is elevated, e.g., in diabetic subjects orpatients with hypercholesterolaemia or hypertriglyceridaemia, theseparticles can become the major lipoprotein fraction responsible forbinding TGF-beta.

Diabetic individuals, particularly those with poor glucose control,often exhibit elevated plasma concentrations of the triglyceride-richlipoprotein particles. Such individuals may therefore have an increasedfraction of their plasma TGF-beta associated with the lipoprotein pool,since they may have a major fraction of their plasma TGF-beta associatedwith the triglyceride-rich lipoprotein particles as well as thesubfraction of HDL particles.

The proportion of TGF-beta in the lipoprotein fraction for ten diabeticindividuals who exhibited poor glucose control was determined(Haemoglobin a1C>8.0). These individuals had moderately elevated totalplasma triglyceride levels (2.34±0.70 mM compared to 1.43±0.60 mM inhealthy control donors; n=10; p<0.07 Student unpaired t-test), and theproportion of TGF-beta associated with lipoprotein was markedlyincreased (68±21% compared to 16±11% in healthy control donors;mean±standard deviation; n=10; p<0.05 Mann-Whitney unpaired U-test).Therefore, diabetic individuals with poor glucose control havesignificantly more of the plasma TGF-beta sequestered into thelipoprotein pool where it is less active or inactive.

EXAMPLE II Effect of Dietary Fish Oil on the Association of TGF-betawith Lipoprotein

To determine whether dietary supplementation with fish oil would reducethe association of plasma TGF-beta with the lipoprotein fraction,platelet-poor plasma was prepared from 33 donors prior to, andimmediately following, four weeks of dietary supplementation with 2.4g/day fish oil (Wallace et al., Arterial Thromb. Vasc. Biol., 15, 185(1995)). A further plasma sample was prepared nine weeks after ceasingthe supplementation. The fraction of TGF-beta associated with thelipoprotein pool was determined for each plasma sample.

At the end of the four week supplementation period total plasmatriglyceride concentrations were somewhat reduced although total plasmacholesterol was unaffected (FIG. 4; Table 2). Fish oil supplementationalso markedly reduced TGF-beta association with the lipoproteinfraction. The mean proportion of TGF-beta associated with lipoproteinwas reduced from 19±10% (range <1% to 62%) to 7±4% (range <1% to 41%;p<0.01; paired Wilcoxon signed-rank test). After a further nine weekswithout fish oil supplementation of the diet, triglycerides had returnedto baseline and the proportion of TGF-beta associated with thelipoprotein pool had increased to 13±9%, although it had not returned tothe baseline.

Consistent with the decrease in the fraction of TGF-beta sequesteredinto the inactive lipoprotein-associated pool, the concentration ofactive TGF-beta increased by 21% after four weeks of dietarysupplementation with 2.4 g/day fish oil. The concentration of activeTGF-beta was still significantly above baseline after a further 9 weeksafter dietary supplementation, although the increase was less marked(+12%, p<0.05). Thus, increased dietary intake of fish oil reduces thefraction of plasma TGF-beta sequestered into the lipoprotein pool, andincreases the concentration of active TGF-beta in plasma.

The reduction in sequestration may be due to the alteration of theproportion of different lipoproteins, i.e., fish oil reducestriglyceride rich lipoprotein levels, or by altering the composition andhence sequestering properties of lipoprotein. Thus, fish oil has noeffect on the production of latent TGF-beta or mature TGF-beta butincreases TGF-beta bioavailability by decreasing the lipoproteinsequestration of the TGF-beta. Such an effect would likely result incardioprotection in individuals with adequate production of latent andmature TGF-beta but limited ability to release TGF-beta from lipoproteincomplexes.

TABLE 2 Time Total Total associated Fish oil triglyceride cholesterol %(weeks) supplementation (mM) (mM) TGF-beta 0 None 1.43 ± 0.43 5.1 ± 1.219 ± 10  n = 32 4 2.4 g/day 1.03 ± 0.57 5.3 ± 0.9  7 ± 4*  n = 33 13 None 1.56 ± 0.50 5.3 ± 0.8 13 ± 9   n = 31 Table 2. Proportion ofTGF-beta associated with lipid following dietary supplimentation withfish oil. Total triglyceride concentration was measured by the glycerolkinase enzymatic method (Sigma Diagnostics). Total cholesterol and %associated TGF-beta were assayed as described in Example I. Values aremean ± standard error. *= p < 0.01; paired Wilcoxon signed-rank testversus baseline.

EXAMPLE III Aspirin Increases Circulating TGF-beta Levels

Aspirin has been suggested to have cardioprotective effects and is nowin widespread use by patients diagnosed with coronary atherosclerosis.It has been demonstrated to significantly reduce the incidence of asecond myocardial infarction (MI) in individuals who have previouslysuffered an MI. However, any benefit for the primary prevention of MIhas not yet been demonstrated rigorously, although some studies havereported encouraging results.

A number of effects have been suggested to play a role in thecardioprotective benefits associated with chronic use of low-doseaspirin. Aspirin interferes with normal platelet function and increasesthe blood clotting time, while decreasing the stability of fibrindeposits. Since chronic formation of mural thrombi is thought to beimportant in the development of atherosclerosis and acute thrombusformation is the main cause of MI, the anti-platelet function of aspirinis thought to be important in mediating its cardioprotective effects.Moreover, since aspirin is a well-documented anti-inflammatory agent andatherosclerosis has an important inflammatory component, theanti-inflammatory action of aspirin could also contribute tocardioprotection.

In a study of 31 individuals with no detectable atherosclerosis bycoronary angiography (NCA), the concentration of active plusacid-activatable latent (a+1) TGF-beta in serum was almost two-foldhigher in those taking aspirin (300 mg per day for an average of 30months) than those not. Thus the proportion of TGF-beta in the activeform was not significantly altered, suggesting that aspirin maystimulate production of the latent TGF-beta precursor rather thanstimulating its activation.

Agents associated with elevated circulating TGF-beta concentration invivo have been shown to stimulate TGF-beta production by vascular smoothmuscle cells (VSMCs) in culture. To determine whether aspirin couldstimulate TGF-beta production by human VSMCs in culture, confluentcultures of human explant-derived VSMCs were subcultured into and grownfor 24 hours in the presence of 10% FCS. The medium was then changed andtriplicate wells were treated with either aspirin (from a stock solutiondissolved in ethanol) or sodium aspirinate at various concentrations.The medium was replaced after 48 hours and after 96 hours the cells werereleased with trypsin and counted by haemocytometer. Tamoxifen (5 μM)was used as positive control, since it has previously been shown tostimulate TGF-beta production under similar conditions. Aspirininhibited the proliferation of human VSMCs with half-maximal inhibition(ED₅₀) at 12±3 μM (n=4), and maximally inhibited proliferation at 50 μMwhen the increase in cell number over 96 hours was inhibited by 33±6%(FIG. 5A). The effects of sodium aspirinate were not distinguishablefrom the effects of aspirin (ED₅₀=10 μM; n=2).

To demonstrate that the inhibition of proliferation by aspirin was dueto TGF-beta production, subcultured human VSMCs were treated with 10 μMaspirin in the presence and absence of 25 μg/ml of a neutralizingantibody to TGF-beta.

The presence of the antibody abolished (>95%; n=3) the growth inhibitoryeffects of aspirin and sodium aspirinate (>95%; n=2) (FIG. 5B).

The amount of TGF-beta present in medium conditioned on VSMCs in thepresence and absence of aspirin was measured by ELISA. In the absence ofaspirin, only 1.5±10.4 ng/ml TGF-beta was detected in the mediumcompared with 4.9±1.2 ng/ml (n=3; p<0.05; Students unpaired t test)after 96 hours in the presence of 10 μM aspirin. Thus, aspirin, liketamoxifen, stimulates production of TGF-beta by human VSMCs in culture,although the ED₅₀ for aspirin (12 μM) was markedly less potent than fortamoxifen (50 nM).

To evaluate the effect of aspirin administration on TGF-beta levels invivo, the level of (a+1) TGF-beta or TGF-beta activity in thecirculation of 42 patients with more than 50% stenoses of all threemajor coronary arteries (TVD) taking low-dose aspirin relative toindividuals with normal coronary arteries (NCA) was determined.Platelet-poor plasma was prepared with minimal (<0.1% assessed by PF-4release) platelet degranulation and active and (a+1) TGF-beta weremeasured by ELISA.

The individuals in the NCA group had (a+1) TGF-beta and active TGF-betalevels typical of healthy individuals reported previously. Thesepatients had either taken no aspirin (n=19), or 75 mg (n=14), 150 mg(n=8) or 300 mg (n=1) of aspirin per day for an average of 17 months.There was a significant correlation between aspirin dose and (a+1)TGF-beta levels (p <0.05; one way analysis of variation) suggesting thataspirin stimulates TGF-beta production in a dose-dependent manner (FIGS.6A and 6B). The mean (a+1) TGF-beta level was significantly higher inpatients taking 75 mg/day of aspirin (+41%; p<0.05), and in patientstaking 150 mg/day of aspirin (55%; p<0.05). This is consistent with aprevious study, where (a+1) TGF-beta levels were elevated by 66% inpatients taking 150 mg aspirin per day. TGF-beta activity was alsoelevated in NCA individuals taking 150 mg aspirin per day (FIG. 6A), andhence the proportion of TGF-beta in the active form was notsignificantly changed. TGF-beta production was similarly higher in bothmen and women taking aspirin (+47% in men compared to 44% in women at150 mg per day; FIG. 6C).

EXAMPLE IV Copper Aspirinate is a TGF-beta Stimulating Agent

One disadvantage of aspirin as a TGF-beta stimulator is that aspirin isnot very potent in human cell culture or in vivo. Therefore, theidentification of other TGF-beta production stimulators which are morepotent than aspirin is needed.

Consumption of red wine has been proposed to mediate cardiovascularprotection, although the data supporting this proposal are stilldebated. To determine whether red wine, as opposed to white wine, canstimulate TGF-beta production in vitro or in vivo, red wine (Chateaux1993 from the Bordeaux region) or white wine (German Reisling) waslyophilized and reconstituted in one tenth volume of 5% ethanol in waterto produce a 10 fold wine concentrate. Red and white grape juice(Sainsbury's) were treated similarly as controls as they are expected tolack the active components produced during fermentation of the grapeskins. Rat vascular smooth muscle cells (rVSMCs) were subcultured intoDMEM+10% fetal calf serum, grown for 24 hours then treated with variousconcentrations of the wines or grape juices. The medium was replacedafter 48 hours and after 96 hours, the cells released with trypsin andcounted by haemocytometer. Final concentrations of the wine and grapejuice on the cells were expressed as a percentage of the concentrationof the original wine or grape juice.

Red wine, but not white wine or either grape juice, inhibited VSMCproliferation with an ED₅₀ of 25-33% concentration. At the highestconcentration tested (200%) the increase in cell number after 96 hourswas inhibited by 46±6% (n=3).

To determine whether this inhibition of VSMC proliferation by the redwine concentrate was due to induction of TGF-beta, cells were incubatedwith 25% and 100% concentration of red wine in the presence and absenceof 10 μg/ml of a neutralizing antiserum to TGF-beta, which haspreviously been shown to completely abolish the growth inhibitoryeffects of 10 ng/ml TGF-beta in VSMC culture. The presence ofneutralizing antibody to TGF-beta completely reversed (>95%) the growthinhibitory effects of the red wine. Thus, red wine induces TGF-betaproduction by VSMCs in vitro and this effect is not due to the alcoholcomponent.

To investigate whether red wine might also elevate TGF-beta levels invivo, blood samples were drawn from 120 randomly selected individuals inToulouse, France and serum prepared. Additionally, these subjectscompleted a questionnaire which included details of their wineconsumption. Active plus latent (a+1) TGF-beta and active TGF-betalevels in these samples were assayed by ELISA as described hereinabove.The mean (a+1) TGF-beta and active TGF-beta levels were notsignificantly different from those reported from other randompopulations.

A weaker correlation was observed between red wine consumption andactive TGF-beta levels. Thus, (a+1) TGF-beta was almost two fold higherin the group of individuals drinking more than 1 standard deviationabove the mean red wine consumption than those more than 1 standarddeviation below the mean. Although there was also a significantcorrelation between total alcohol consumption and (a+1) TGF-beta levels,this may result from the large fraction of total alcohol consumed whichis taken as red wine in this population. There was no correlationbetween white wine, beer or spirit consumption and either (a+1) TGF-betaor active TGF-beta levels. Thus, it is very likely that increased wineconsumption is associated with elevated TGF-beta levels in thecirculation.

When the red wine consumed was divided by the region of origin, fourregions were significantly represented. However of these, onlyindividuals drinking wine originally from Bordeaux showed astatistically significant correlation with (a+1) TGF-beta levels. Thismay be a consequence of the reduced numbers of individuals in eachgroup, or alternatively suggests that only wines of Bordeaux originstimulate TGF-beta production or increase TGF-beta levels more potentlythan wines of other origins.

Red wines, but not white wines, have been shown to contain varioussalicylate components which are produced during fermentation of thegrape skins. To determine whether a salicylate component of red wine wascorrelated to TGF-beta activity, cultured (rat or human) VSMCs wereexposed to red wine in the presence of various concentrations of aneutralizing antibody raised against sodium salicylate coupled tokeyhole limpit hemocyanin as a carrier protein. The anti-salicylateantibody reversed the growth inhibitory activity of Bordeaux red winewith an ED₅₀ of 15 μg/ml. At concentrations of 33 μg/ml and above,maximal reversal of the growth inhibition was achieved, i.e.,approximately 70% of the growth inhibitory activity was reversed. Themajority of the TGF-beta stimulating activity in Bordeaux red wine istherefore due to the presence of salicylate-like compounds.

Given the likely concentration of salicylate in this red wine, based onprevious studies, and the ED₅₀ for aspirin inducing TGF-beta in VSMCculture, the presence of salicylate alone cannot explain the observedeffects. One possible resolution of this paradox would be the presenceof salicylate-like compounds in red wine which stimulate TGF-beta morepotently than acetyl salicylate. One such derivative, reported to havemore potent effects than aspirin is the transition metal complex copperII (acetyl salicylate)₂. The ED₅₀ for TGF-beta production of the complex(Cu Aspirinate) was determined in cultured rat and human VSMCs. Cuaspirinate was almost two orders of magnitude more potent than aspirinat stimulating TGF-beta (ED₅₀ on human cells 200 nM for Cu aspirinateversus 10 μM for aspirin). It is likely that there is sufficient Cuaspirinate in red wine, and particularly in red wines of Bordeaux originwhich are especially rich in copper, to account for most if not all ofthe TGF-beta stimulating activity associated with red wine.

Thus, copper aspirinate complex is believed to be the active TGF-betastimulating agent in red wine and is a potent TGF-beta productionstimulating agent in vitro and in vivo.

EXAMPLE V TGF-Beta Levels in Tamoxifen Treated Patients

To investigate whether TGF-beta levels are elevated after TMXadministration, fifteen patients with stable angina and angiographicallydefined triple vessel disease took TMX daily for ten days at a dosesimilar to that generally used for breast cancer therapy. Patients withtriple vessel disease (TVD) were defined as individuals having at least50% stenosis of all three coronary arteries by angiography, which wasconfirmed by two independent observers. All had stable angina, with nomyocardial infarction in the previous three months. Patients withunstable angina, poor left ventricular function, ventricular hypertrophyor diabetes were excluded.

Blood samples were taken and plasma prepared before and during thetreatment period, and these samples were analyzed for TGF-beta, Lp(a),PAI-1 and lipoprotein profiles. Patients were asked to fast overnightprior to samples of blood being drawn between 9 a.m. and 10 a.m. thefollowing morning. Blood samples were drawn by venepuncture of theantecubital vein with no tourniquet applied using a 21 gauge butterflyneedle. Half the blood was allowed to clot for 2 hours at roomtemperature in polypropylene tubes. The clot was spun down (1,500× g; 15minutes) and aliquots of the serum was stored at −100° C. The remainingblood was dispensed into Diatube H tubes (Diagnostica Stago) and cooledon ice for 15 minutes. Blood cells and platelets were spun down (6,000×g; 30 minutes) and the middle third of the supernatant was taken,carefully avoiding disturbing the pellet. This platelet-poor plasma wasstored in aliquots at −100° C. until assayed. For all samples, assay forPF-4 demonstrated that less than 0.02% of the platelets had degranulatedduring plasma preparation.

Active plus acid-activatable latent (a+1) TGF-beta levels were assayedin platelet-poor plasma using seven different assay methods. There hasbeen debate in the literature regarding the most appropriate way tomeasure (a+1) TGF-beta, so to avoid difficulties specific to anyparticular measurement method all the available methods which have beendescribed in the literature were used. The seven methods are:

(A) A sandwhich ELISA using BDA19 and BDA5 (R&D Systems) antibodies withno activation step required.

(B) The Quantikine TGF-beta1 ELISA kit (R&D Systems) using acid/urea asthe activation buffer in accordance with the manufacturer's instructions

(C) The Quantikine TGF-beta1 ELISA kit (R&D Systems) using acid alone asthe activation buffer

(D) The BioTrak TGF-beta1 ELISA kit (Amersham International) usingacid/urea as the activation buffer

(E) The BioTrak TGF-beta1 ELISA kit (Amersham International) using acidalone as the activation buffer in accordance with the manufacturersinstructions

(F) The TGF-beta1 ELISA kit (Genzyme Diagnostics) using acid/urea as theactivation buffer in accordance with the manufacturer's instructions

(G) The TGF-beta1 ELISA kit (Promega Corporation) using acid alone asthe activation buffer in accordance with the manufacturer's instructions

Replicate aliquots of plasma taken from the same individuals at the sametime were assayed by all seven methods.

The partitioning of TGF-beta between the lipoproteins and plasmaproteins was analyzed by separating the lipoprotein fraction (d<1.215g/cm²) from the plasma proteins by density ultracentrifugation asdescribed hereinabove. TGF-beta levels were assayed in both fractionsusing the Quantikine ELISA kit, following release and activation ofTGF-beta with acetic acid and urea in accordance with the manufacturer'sinstructions. None of the three TGF-beta ELISAs used here detectedTGF-beta in the lipoprotein fraction without prior extraction/activationwith acetic acid/urea.

Total plasma triglyceride, total plasma cholesterol, HDL-cholesterol,LDL-cholesterol and VLDL-cholesterol were routinely assayed in allpatients. Liver function tests (aspartate transaminase and lactatedehydrogenase) were also performed on samples prior to dosing with TMXand at the end of the study by a clinical biochemistry laboratory.Plasma PAI-1 was assayed using an ELISA (American Diagnostica) whichrecognizes active endothelial PAI-1 as well as inactive PA/PAI-1complexes. Lipoprotein(a) was assayed by an ELISA for apolipoprotein(a)(immuno) which showed no detectable cross reactivity with relatedproteins such as plasminogen. Platelet factor-4 (PF4) andβ-thromboglobulin (βTG) were assayed using specific ELISAs (DiagnosticaStago).

Many of the parameters studied would not be expected to show a normaldistribution in the population of TVD patients. Consequently, allcomparisons are made with the baseline (day 0) values using the pairedWilcoxon signed-rank test. A p value of 0.05 or less was taken toindicate statistical significance.

TMX [Nolvadex™ (tamoxifen citrate), Zeneca Ltd., Macclesfield, UK] at adose of 40 mg was taken each morning, before breakfast, for 10 days.Before TMX treatment the mean (a+1) TGF-beta in plasma was 6.2±1.3ng/ml.

During treatment with TMX, there was a trend of increasing concentrationof (a+1) TGF-beta irrespective of the assay method used (Table 3). Eachof the assay methods were standardized against different TGF-betastandard curves and gave significantly different median levels of (a+1)TGF-beta in the population at baseline. However, by day 10 there was amedian increase of 59% in (a+1) TGF-beta. This increase wasstatistically significant for all the assay methodologies used, exceptfor (G). This kit did not perform well, detecting much lower levels of(a+1) TGF-beta at both baseline and after treatment than all the othermethods (A) to (F).

Therefore, during treatment of men with TMX there is a statisticallysignificant increase in the amount of (a+1) TGF-beta in plasma, by 10days after treatment is commenced. This increase is detectedirrespective of the methodology used to measure (a+1) TGF-beta.

TABLE 3 Day 0 Day 10 Age (yrs) 62.2 ± 1.5  Total plasma cholesterol 6.31± 0.28  5.95 ± 0.29* (mM) VLDL-cholesterol (mM) 1.03 ± 0.14  0.84 ±0.11* LDL-cholesterol (mM) 4.48 ± 0.27 4.16 ± 0.25 HDL-cholesterol (mM)0.78 ± 0.03 0.77 ± 0.04 Total plasma triglycerides 2.79 ± 0.44 2.28 ±0.35 (mM) Plasma (a + 1) TGF-β (ng/ml) Method (A) 6.2 ± 1.3  7.7 ± 1.5*(+24%) Method (B) 0.7 ± 0.1  1.2 ± 0.2* (+71%) Method (C) 0.25 ± 0.07 0.62 ± 0.07** (+148%)  Method (D)  2.0 ± 0.1*  2.4 ± 0.1* (+20%) Method(E) 1.7 ± 0.1  2.3 ± 0.1** (+29%) Method (F) 3.9 ± 0.1  5.2 ± 0.3**(+59%) Method (G) 0.1 ± 0.1 0.2 ± 0.1 (+100%)  Lipoprotein(a) (mg/dl)61.3 ± 13.8  42.4 ± 9.5** Plasma PAI-I antigen 29.3 ± 6.4  35.9 ± 5.4*(ng/ml) All values are mean ± standard error for 15 patents. Comparisonsbetween baseline (day 0) and values after TMX treatment (day 10) weremade using the paired Wilcoxon signed-ranks test. *, p < 0.05; **, p <0.01.

These patients had a high average level of Lp(a) (mean 61±10 mg/dl atbaseline), consistent with their coronary artery status. The plasmaconcentration of Lp(a) was decreased by 27% (p 0.05 compared tobaseline) by day 3 of TMX therapy. By day 10, the Lp(a) concentrationwas reduced by 31% compared to baseline (p=0.02; FIG. 8A).

Another cardiovascular risk factor which has been shown to influenceTGF-beta activity is the lipoprotein profile, since TGF-beta can besequestered into lipoprotein particles where it is biologicallyinactive. TMX has been reported to decrease plasma cholesterol and toincrease the fraction of cholesterol in HDL particles. Consistent withthese reports, total plasma cholesterol was decreased by 6% belowbaseline (p=0.04) after 10 days of TMX therapy. In addition, cholesterolin the VLDL fraction was reduced (18% below baseline; p=0.04) but theconcentration of LDL-cholesterol and HDL-cholesterol were both unchanged(Table 3). Total plasma triglyceride concentration was 18% lower after10 days of TMX treatment, but the change was not statisticallysignificant (p=0.22).

Since TMX had significantly altered the lipoprotein profile of thepatients, the proportion of the plasma TGF-beta associated withlipoprotein was measured. The lipoproteins were separated from theplasma proteins by density gradient ultracentrifugation. In order todetect the TGF-beta1 in the lipoprotein fraction, the Quantikine ELISAwas used following release and activation of any TGF-beta in both thelipoprotein fraction and the plasma protein fraction. At baseline 34±4%of the TGF-beta was lipoprotein-associated and hence biologicallyinactive, but this was reduced to 25±3% (p<0.01) after 10 days of TMXtherapy (FIG. 8C).

The data show that TMX (40 mg per day) elevates the plasma concentrationof TGF-beta in men with severe coronary atherosclerosis. This increasewas seen irrespective of which of the seven different methodologies wereemployed to measure (a+1) TGF-beta. Consistent with studies in cellculture and in mice, TMX elevates the amount of (a+1) TGF-beta,suggesting that the elevation may have resulted from increased synthesisof latent precursor complexes. In rat and human smooth muscle cellculture, TMX increases TGF-beta production by increasing the amount ofTGF-beta1 mRNA. In other cell types TMX increases the translationalefficiency of TGF-beta mRNA and hence increases production of the latentprecursor protein. Irrespective of the mechanism, we observe increasedlevels of TGF-beta in men with atherosclerosis, corresponding to theincreases seen in animal models of atherosclerosis when TMXsignificantly reduces lipid lesion formation, irrespective of thegenetic predisposition to lesion formation (FIG. 9).

EXAMPLE VI Combination Therapies to Elevate the Level of TGF-Beta

Another disadvantage of aspirin as a cardiovascular agent, besides thefact that it is not a very potent TGF-beta elevating agent, is that itappears to be a pure stimulator of the latent form of TGF-beta. As aresult, under conditions where TGF-beta activation or release is notoccurring, or is occurring to a reduced extent, e.g., when PAI-1inhibits activation or lipoproteins sequester TGF-beta, the supply oflatent TGF-beta precursors may not be limiting for the generation of theactive forms. This disadvantage can be overcome by combination therapy.Thus, the identification of agents that increase the level of matureand/or active TGF-beta, can be useful in combination therapies withaspirin or with other agents that are more potent stimulators of thelatent form of TGF-beta, such as copper aspirinate.

To determine the efficacy of combination therapy, and to provideevidence for synergism between aspirin and fish oil, 8-week-old femaleapoE knockout mice were fed aspirin or fish oil, or both, to assess thecardioprotective effects of modulating different components of theTGF-beta pathway.

Group A mice (n=10) were sacrificed at day 0. Group B mice (n=10) werefed normal chow. Group C mice (n=10) were fed normal chow and about 3mg/kg/day aspirin dissolved in water (15 μg/ml aspirin). Group D mice(n=10) were fed chow containing 33 mg/kg/day fish oil (200 μg Pulse codliver oil/g food, Seven Seas Ltd., which contains 0.9 g eicosapentaenoicacid (EPA), and 0.3 g docosahexaenoic acid (DHA)) and 3 mg/kg/dayaspirin dissolved in water. Group E mice (n=10) were fed chow containing33 mg/kg/day fish oil. Group F mice (n=10) were fed chow containingZocor (simvastatin; Zocor tablets, Merck, Sharpe & Dohme) at 400μg/kg/day (2 μg/g food). Simvastatin is an inhibitor of the enzymeHMG-CoA reductase, the committed step in cholesterol biosynthesis. As aresult, it has been shown to reduce the total plasma cholesterolconcentration in man and in particular the concentration of cholesterolin the more triglyceride-rich particles (VLDL and LDL). If alterationsin the lipid profile are responsible for the suppression of lesionformation previously observed with TMX, then simvastatin should reducelesion formation.

Groups B-F were fed these regimens for 87 days. All mice were weigheddaily for the first week and weekly thereafter. Food and waterconsumption over a 24 hour period were measured daily for the first 7days and weekly thereafter. There was no significant difference inweight, food intake or water consumption in any of the groups throughoutthe study.

After 87 days, mice in groups B-F were fasted overnight and thensacrificed. Serum, heart, lungs and aorta samples were collected at thetime of sacrifice. The heart, lungs and aorta were removed from eachmouse and rinsed in PBS, dabbed dry on tissue and embedded in Cryo-M-bedembedding medium (Bright Instruments, Huntington, U.K.) before snapfreezing in liquid nitrogen. Frozen sections (4 μm thickness) of theaortic sinus region were prepared from the heart/lung/aorta blocksaccording to the sectioning strategy of Paigen et al. (Arteriosclerosis,10, 316 (1990)). Sections on 5% gelatin-coated slides were stained forneutral lipid by the Oil Red O technique and counter-stained with fastgreen (Grainger et al., Nature Med., 1, 1067 (1995)).

The development of lipid-filled vascular lesions was determined by thequantitation of oil red O staining for neutral lipid deposited in theaortic sinus region. The area of lipid accumulation was measured using acalibrated microscope eye-piece, such that lipid droplets <50 μm² wereignored and contiguous regions of lipid staining >500 μm² in area wereclassified as lesions. The area staining for neutral lipid increasedfrom 10,765±978 to 27,175±1040 μm²/mouse over the three months of theexperiment for mice fed a normal mouse chow diet, as seen in previousstudies of spontaneous lesion development in apoE knockout mice.Treatment with aspirin alone did not affect lesion development over thesame 3 month period (Table 4). Treatment with fish oil alone reducedlesion development slightly, although the variation in the area oflesions between animals within a group was too large for the effect tobe statistically significant (−8%; p=0.11; Mann-Whitney U test).

In contrast, treatment with aspirin plus fish oil resulted in asignificant reduction in lesion formation (−22%; p=0.01; Mann-Whitney Utest), suggesting that aspirin and fish oil act synergistically toreduce lipid lesion formation. Treatment with simvastatin, however, didnot significantly reduce lipid lesion formation in apoE knockout mice.The area staining for neutral lipid deposition was lower than inuntreated mice (−7%; p=0.33; Mann-Whitney U test), but as with micetreated with fish oil alone, this decrease was not statisticallysignificant.

Treatment with aspirin and fish oil, alone or in combination, alsoresulted in a marked change in lesion morphology. The area of cellularintima that formed was reduced, most markedly in the group whichreceived the combination of aspirin and fish oil, and the lipid stainingwas confined to a region close to the internal elastic lamina. As aresult, the lesions coalesced and the number of separate lesionsdecreased even where the total area staining for lipid accumulation wasunchanged.

TABLE 4 Lesion Area Group Treatment (μm² staining) Number of Lesions Acontrol, day 0 10,765 ± 978  3.4 ± 0.2  B control, day 88 27,175 ± 104010.5 ± 0.7   C aspirin 27,512 ± 974  6.6 ± 0.2** D aspirin + fish oil 23,587 ± 898** 5.5 ± 0.4** E fish oil 25,871 ± 1356 6.9 ± 0.3** F Zocor25,777 ± 1368 8.1 ± 0.4*  *p < 0.01, **p < 0.05 Mann-Whitney U test

The amount of active plus latent and active TGF-beta in the vessel wall,and the amount of active TGF-beta (ng/ml) in the plasma of these micewas also determined (Table 5), by methodologies described in Examples 4and 7 of copending U.S. application Ser. No. 08/478,936, filed Jun. 7,1995, which is incorporated by reference herein.

The neighboring sections to those stained for neutral lipid with Oil Red0 described above were taken onto slides coated with poly-L-lysine(0.1%; Sigma) and fixed in ice-cold acetone for 90 seconds, air-driedand stored at −20° C. until assayed. Active plus acid-activatable latent(a+1) TGF-beta was measured by quantitative immunofluorescencemicroscopy using specific primary antibodies (BDA 19, AB-100-NA; R&DSystems), see Mosedale et al., Histochem. Cytochem., 44, 1043 (1996),the disclosure of which is incorporated by reference herein. ActiveTGF-beta was measured by quantitative immunofluorescence microscopyusing the recombinant extracellular domain of the type II TGF-betareceptor (R2X) labeled with fluorescein.

The data in Table 5 show that active plus latent TGF-beta levels weresignificantly elevated in the vessel wall of mice having dietssupplemented with aspirin (+36%; p<0.01; n=10, Mann-Whitney U test)relative to control mice. The amount of active TGF-beta was notsignificantly affected by aspirin therapy (−6%; p=NS), suggesting thatthe additional latent complexes were not efficiently activated in thevessel wall of apoE knockout mice. In contrast, fish oil treatment forthree months did not affect (a+1) TGF-beta (+5%; p=NS) but elevatedactive TGF-beta to a small extent (+17%; p=0.05; n=10; Mann-Whitney Utest). These results suggest that aspirin stimulates production oflatent TGF-beta complexes, while fish oil increases the proportion ofTGF-beta available in the active form, i.e., for receptor binding.

TABLE 5 Active + Latent Active TGF- TGF-beta in beta in Vessel GroupVessel Wall Wall A 54 ± 4 36 ± 2 B 42 ± 4 18 ± 2 C  57 ± 3** 17 ± 2 D 63 ± 6**  24 ± 3** E 44 ± 4  21 ± 3* F 44 ± 5 20 ± 3 *p < 0.01, **p <0.05 Mann-Whitney U test

Group D mice, which were treated with both aspirin and fish oil hadsignificantly elevated levels of both (a+1) TGF-beta (+50%) and activeTGF-beta (+33%) in the vessel wall compared with the control mice. Thesynergism of the effects of these drugs on the amount of active TGF-betain the vessel wall is consistent with the proposed different mechanismsof action for the two drugs. Taken with the results shown in Table 4, anincrease in level of active TGF-beta in apo(E)−/− mice correlates with adecrease in lesion number and area.

Simvastatin treated mice (Group F) showed no difference in the amountsof (a+1) TGF-beta and active TGF-beta in the vessel wall. Thus, in theapoE knockout mouse, any beneficial effects of simvastatin are unlikelyto be attributed to elevation of TGF-beta activity. Similarly, anybeneficial effects of the aspirin plus fish oil therapy on thelipoprotein profile are unlikely to have contributed to the therapeuticreduction in lesion area by this therapy.

In summary, agents that elevate TGF-beta activity in the vessel wallreduce or inhibit lipid lesion development in mouse aorta, while agentswhich do not affect TGF-beta activity are ineffective (Table 6).Furthermore, the statistical correlation between the magnitude ofTGF-beta activity elevation and lesion area inhibition is very marked,suggesting that the greater increase in vessel wall TGF-beta activitywhich is achieved, the greater the inhibition of lesion development.This correlation provides powerful evidence supporting the role ofTGF-beta activity in mediating the cardioprotective activity of bothtamoxifen, and aspirin and fish oil.

TABLE 6 Fold increase in Active Treatment % Lesion Suppression TGF-betaNone 0 1.00 Aspirin −2   0.95 Fish Oil 8 1.1 Aspirin and Fish Oil 17*1.3* TMX 99* 1.9* *Statistically significant, p < 0.001, Pearson's Rcorrelation, r = 0.73

The effects of these treatments on the cellular changes associated withlesion formation, marked by the accumulation of osteopontin and loss ofsmooth muscle α-actin (SM-α-actin) in the vessel wall, which has beenshown to be characteristic of lesion formation in man and animal modelsof atherosclerosis, was also examined. SM-α-actin and osteopontin weremeasured by quantitative immunofluorescence microscopy using specificprimary antibodies A-6125 (Sigma) and MBP111 Bio (NIH DevelopmentalStudies Hybridoma Bank), respectively.

As lipid lesions developed over 3 months on a normal mouse chow diet,staining for SM-α-actin decreased (−36%; p<0.01; n=10; Mann-Whitney Utest), while staining for osteopontin increased (+150%; p<0.01; n=10;Mann-Whitney U test). Of the treatments used in this study, only thecombination of fish oil and aspirin abolished the loss of SM-α-actin andthe accumulation of osteopontin (Table 7), consistent with theobservation that this was the only treatment regimen which significantlyreduced lipid accumulation into the vessel wall. The increase in SMαactin in mice treated with fish oil or fish oil plus aspirin isconsistent with the observed increase in SMα actin in apo(E)−/− micetreated with TMX.

TABLE 7 Group SMα Actin Osteopontin A 157 ± 22 32 ± 7 B 101 ± 12 80 ± 9C  87 ± 9* 84 ± 7 D  194 ± 18**  55 ± 8** E 122 ± 17  67 ± 10* F 114 ±19 73 ± 8 *p < 0.01, **p < 0.05 Mann-Whitney U test

TABLE 8 Active & Latent Active TGF-beta TGF-beta SMα Actin Oil Red OActive & latent r = 0.58** r = 0.67*** r = −0.065 TGF-beta Active TGF- r= 0.76*** r = −0.32* beta SMα actin r = −0.13 Oil Red O *p < 0.01 **p <0.001 ***p < 0.0001

As shown by the correlations of data from Groups A-F summarized in Table8, an increase in the level of active TGF-beta correlates with anincrease in SMα actin expression. This result is consistent with thehypothesis that active TGF-beta regulates smooth muscle celldifferentiation in vivo. Moreover, active TGF-beta, but not active pluslatent TGF-beta, negatively correlates with lesion area, suggesting thatactive TGF-beta protects against lesion development.

The effect of each treatment on the lipid profile of each group of micewas determined by measuring the cholesterol and triglyceride. Blood froma terminal bleed was collected in a polypropylene tube, allowed to clotat room temperature for 2 hours and then spun (1,000× g; 5 minutes). Theserum supernatant was aliquoted and stored at −20° C. until assayed.Total cholesterol and total triglycerides were determined for each mouseusing the cholesterol oxidase and glycerol kinase UV end-point enzymaticmethods respectively (Sigma Diagnostics). For determination of thelipoprotein profile, 100 μl of serum from every mouse in each group waspooled (a total of 1 ml serum for each group) and the lipoproteinfraction was separated by density gradient ultracentrifugation. Thelipoprotein fraction was then further separated by gel filtration FPLCchromatography on a Sepharose 6B column, and the elution positions ofthe lipoprotein particles were detected by measuring cholesterol (by thecholesterol oxidase enzymatic method) in each fraction. VLDL particleseluted in fractions 1-10, LDL in fractions 11-20 and HDL in fractionsafter 20.

Treatment of the mice with aspirin for three months had no effect ontotal plasma cholesterol or on the lipoprotein profile (Table 8). Micetreated with diets containing fish oil (with or without aspirin) hadsimilar total plasma cholesterol and triglyceride concentrations tocontrol mice, although there was a small reduction in the concentrationof both VLDL-cholesterol (−16%) and LDL-cholesterol (−12%) and anincrease in HDL-cholesterol (+10%). Consistent with the effects ofdietary supplementation with fish oil in man, a decrease in cholesterol,primarily in the VLDL fraction, in apoE knockout mice treated with fishoil was observed.

There was a significant reduction in total plasma cholesterol in apoEknockout mice treated with simvastatin (−27%; p<0.01; n=10; Studentsunpaired t-test). Much of this reduction occurred in the VLDL fraction(−14%) and LDL fraction (−41%), with an increase in HDL-cholesterol. Incontrast, TMX lowered VLDL by seven fold and is a much more powerfullipid-lowering agent in the apo(E)−/− mouse than simvastatin.Simvastatin also caused a significant reduction in total plasmatriglyceride concentration (−12%). Consequently, for all lipoproteinparameters measured, simvastatin had a significantly more beneficialeffect than aspirin and fish oil either alone or in combination.

TABLE 9 Group A Group B Group C Group D Group E Group F Totalcholesterol (mg/dl) n.d. 306 ± 31 282 ± 28 273 ± 19 266 ± 25 224 ± 29**Total triglyceride (mg/dl) n.d. 302 ± 28 320 ± 19 308 ± 25 296 ± 33 266± 14** VLDL-cholesterol (mg/dl) n.d. 184  179  157  151  158 LDL-cholesterol (mg/dl) n.d. 92 89 91 88 54 HDL-cholesterol (mg/dl) n.d.30 26 32 33 35 **p < 0.001; Mann-Whitney U test n.d. = not determined. Asingle measurement of the lipoprotein profile was made on blood pooledfrom all the mice in the Group.

Moreover, the percentage of TGF-beta sequestered in VLDL in Groups B-Fand C57B16 mice, which were fed a high fat diet, showed that lipidsequestration of active TGF-beta was not a major mechanism of theinhibition of TGF-beta activity in apo(E)−/− mice.

In summary, aspirin and fish oil act synergistically to reduce aorticlipid lesion development in a mouse model of severe atherosclerosis.While aspirin or fish oil alone reduced the development of vascularlipid lesions in apoE knockout mice over a three month treatment period,a combination of aspirin plus fish oil therapy resulted in a greaterreduction (22%) in lesion formation. If low dose aspirin therapy anddietary supplementation with fish oil differ in their mechanism ofaction, then their cardioprotective effects would be expected to beadditive. However, the results described hereinabove provide evidencethat the combination of aspirin and fish oil exerts a markedlysynergistic effect. Thus, a combination of low dose aspirin and fish oiltherapy can be very useful in cardiovascular disease prevention.Moreover, because fish oil is not a very effective VLDL lowering agent,more powerful VLDL lowering agents, such as TMX, can be employed incombination therapies with aspirin, aspirinate salts to result in morebeneficial cardiovascular effects.

Consistent with data in humans, aspirin increases the level of latentTGF-beta, but not the amount of active TGF-beta, in the vessel wall ofapo(E)−/− mice. Also consistent with data in humans, fish oil lowersVLDL, which results in lower levels of PAI-1 and an increase in thelevels of active TGF-beta which are available for TGF-beta receptorbinding.

Previously, tamoxifen treatment has been demonstrated to elevateTGF-beta activity and suppress lipid lesion formation in severaltransgenic mouse models of atherosclerosis (Grainger et al.). However,tamoxifen has a variety of other effects, including reducing totalplasma cholesterol and inducing some weight loss, which may havecontributed to the observed reduction in lesion development. As aresult, it could not be concluded that elevating TGF-beta activityreduced lesion formation. In contrast, the study described hereinaboveemployed agents which elevate TGF-beta activity and which do not affectbody weight and have much smaller effects on lipoprotein metabolism.Furthermore, simvastatin, which has a larger beneficial effect on thelipoprotein profile than the other treatments, does not significantlyreduce lipid lesion formation. Since there is a significant correlationbetween increase in TGF-beta activity and decrease in lipid lesionformation for all the therapies (r=0.909; p<0.001), it can be concludedthat elevation in TGF-beta activity is likely to be involved in themechanism by which these agents reduce lesion formation in mammals.

EXAMPLE VII Use of Therapeutic Agents of the Invention to PreventAutoimmune Disorders

The therapeutic agents of the invention are also useful to prevent ortreat other indications associated with TGF-beta, e.g., pathologieswhich result from a pathological inflammation reaction caused by therecognition of self-antigens (“autoimmune disorders”). Indicationsassociated with pathological inflammation reactions include, but are notlimited to, rheumatoid arthritis, multiple sclerosis and late-onsetdiabetes. The recruitment and activation of both autoreactive T cellsand other inflammatory cells to the developing lesion contributes toboth the chronic tissue damage and the acute symptoms of autoimmunedisorders. Agents which reduce or prevent immune cell recruitment and/oractivation may ameliorate both the painful symptoms associated with thedisorder and the progressive destruction of the target tissue.

Current treatments for autoimmune disorders include the administrationof anti-inflammatory steroids and steroid-mimetic drugs, such asdexamethasone, to reduce recruitment and activation of the immune cellsin the developing lesions. These drugs act by binding to theglucocorticoid receptor (GR) which leads to the association of the GRwith elements of the NFkB transcription factor complex. When the GRinteracts with the NFkB complex, pro-inflammatory cytokines areinhibited. The binding of the steroids to the GR also results in theactivation of GR. Activated GR is a nuclear transcription factor. Thus,a set of genes are activated in response to the binding of a steroid orsteroid-mimetic drug to GR. This pathway is illustrated in FIG. 12.However, steroids and steroid-mimetic drugs cannot be used chronicallyto slow the progression of autoimmune diseases because they have anundesirable profile of side effects. Many or all of these side effectsresult from the direct activation of the GR as a transcription factor.

Thus, agents which modulate the interaction of the estrogen receptor(ER) with the NFkB transcriptional complex (“ER/NFkB modulators”)without activating GR are useful to prevent or treat conditionscharacterized by the recruitment of autoreactive immune cells intotissue and the subsequent damage or destruction of that tissue bychronic inflammation. Preferred ER/NFkB modulators include idoxifene,raloxifene, droloxifene, toremifene, and tamoxifen, as well asfunctional equivalents, analogs or derivatives thereof. These agentsalso inhibit or reduce TNF-alpha mediated NFkB activation. Moreover, asER/NFkB modulators are not characterized by the undesirable side effectprofile of GR/NFkB modulators at the doses used to treat autoimmunedisorders, they are therefore amenable to chronic use in the preventionor treatment of autoimmune disorders.

When cells which have NFkB activity, such as human smooth muscle cells(SMCs), were cultured in the presence of 20% fetal calf serum (FCS) inDulbecco's modification of Eagles' Medium (DMEM), more than 95% of NFkBwas present in the cytoplasm, as determined by immunostaining for p65.When the cells were incubated with a pro-inflammatory cytokine(recombinant human TNF-alpha at 20 ng/ml) for 6 hours at 37° C., >80% ofthe NFkB was translocated to the nucleus. In cells which had beentransfected with a reporter construct which has three consensus kB DNAelements fused to the luciferase gene, the amount of light which wasproduced when the cell lysate was exposed to luciferin and ATP wasproportional to NFkB activity. Treatment of human SMCs for 6 hours with20 ng/ml TGF-alpha resulted in a 19-fold increase in NFkB activity,consistent with the translocation of the NFkB complex to the nucleus.

In contrast, when human SMCs were treated with tamoxifen, NFkB remainedin the cytoplasm and there was no detectable change in NFkB activity.When the human SMCs were incubated simultaneously with TNF-alpha (20ng/ml) and tamoxifen (5 μM), less than 10% of the NFkB was translocatedto the nucleus and NFkB activity was stimulated by less than 3 fold. Atthis concentration, tamoxifen (TMX) inhibits TNF-alpha-induced NFkBactivity by 86±12%.

Binding of tritium-labeled tamoxifen (3H—TMX) was used to determine theaffinity and number of binding sites for TMX in total cell lysatesprepared from human VSMCs. Scatchard analysis of the binding of ³H-TMXrevealed the presence of at least 3 distinct binding sites presentat >1000 binding sites per cell. Site A had an affinity of 19 nM and waspresent at 4000 binding sites per cell. Site B had an affinity of 40 nMand was present at 9000 binding sites per cell and site C had anaffinity of 3 μM and was present at 100,000 binding sites per cell.These results are consistent with Site B being the free human ERprotein. It is likely that the higher affinity site A also contained theER as it was efficiently immunoprecipitated by monoclonal antibodiesdirected against the human ER protein.

To identify other targets of TMX, an analog of 4-iodotamoxifen wascovalently coupled to agarose and used to affinity purify antiestrogenbinding proteins from total cell lysates prepared from human SMCs. Boundtamoxifen-binding proteins were eluted with the water-soluble quaternarytamoxifen salt N-methyl tamoxifen iodide. The eluting salt was removedby dialysis against Amberlite resin in phosphate buffer whichirreversibly binds N-methyl tamoxifen. The affinity purified proteinswere separated further by MonoQ ion exchange chromatography andfractions were assayed for ³H-TMX binding. Three peaks of proteinassociated with TMX-binding activity were identified. Peak I had anaffinity of about 1 μM and may correspond to site C. Furtherpurification of this protein by gel filtration chromatography and gelelectrophoresis allowed a molecular identification of the protein byN-terminal sequence analysis as human serum albumin. The amount ofprotein in the other two peaks of activity was less than the amountnecessary to allow molecular characterization of these proteins.

Human SMC lysates were treated with a large excess of antibody directedagainst the human ER protein. After rotating overnight at 4° C., theantibody:antigen complex was precipitated by addition of 20 μl/mlprotein A/G agarose and centrifuged for 5 minutes at 4° C. Thesupernatant was treated similarly, until no further ER protein could bedetected in the precipitated fraction. At concentrations of ³H-TMX below50 nM (when the contribution by the low affinity site C should benegligible), all of the ³H-TMX binding sites were removed by treatmentwith the antibody directed against the human ER. Thus, both sites A andB (the high affinity sites) contain either the human ER or a proteinwhich contains an epitope conserved with the human ER. It is very likelythat both TMX binding complexes contain the ER.

To determine whether any ER protein in human VSMCs was complexed withother proteins, human SMCs cultured in DMEM+20% FCS were grown overnightin methionine-free DMEM+20% dialyzed FCS, then incubated with 50 μCi/ml³⁵S-labeled methionine in methionine-free DMEM+20% dialyzed FCS for 6hours. Total cell lysates were prepared from the labeled cells andimmunoprecipitated with antibodies to ER. The immunoprecipitatedproteins were then analyzed by SDS gel electrophoresis andautoradiography. As expected, a band at 88 kDa corresponding to the ERprotein was detected. Additionally, a band at 92 kDa was detected.Subsequent Western blotting determined that the 92 kDa band was the heatshock protein hsp90, which has been shown to be associated with ER inthe cytoplasm. A third protein was also efficiently immunoprecipitatedwith the ER. This third protein migrated at 37-40 kDa. Since it has beenshown that the GR steroid receptor interacts with NFkB transcriptionfactor complexes, the 37-40 kDa protein was analyzed by Western blottingwith antibodies directed against IkB-alpha. Human IkB-alpha has beenreported to migrate as a 37 kDa protein. These experiments confirmedthat human IkB-alpha forms complexes with human ER either as a ternarycomplex with hsp90 or with human ER alone.

Whole cell lysates from human SMCs were treated with antibody toIkB-alpha until no further IkB-alpha was found in the precipitatedfraction. The supernatant had about 50% of the binding sites for ³H-TMXpresent in the original lysate, while immunoprecipitation withnon-immune antiserum did not reduce the number of TMX binding sites bymore than 5%. Therefore, the three TMX binding sites in human SMC celllysates are human serum albumin, a complex containing ER and IkB-alpha,and a complex containing ER but not IkB-alpha.

Because ER interacts with NFkB transcription factor complexes in asimilar manner to that for GR, agents which modulate ER/NFkB interactionshould modulate the inflammatory response without activating GR. To testthis hypothesis, SMCs in DMEM+10% FCS were transfected with a vectorcomprising the MMTV LTR promoter coupled to the chloramphenicol acetyltransferase (CAT) gene and the neomycin resistance gene (neo). A stablytransfected line was selected using geneticin. When these cells weretreated with dexamethasone, expression of the CAT gene was elevated3.7±0.7 fold. Treatment of these cells with concentrations of TNF-alpha(up to 100 ng/ml), tamoxifen (up to 10 μM) or both agents together, didnot stimulate expression of the CAT gene by more than 10%. Thus, ER/NFkBmodulators would be expected to circumvent the undesirable side-effectprofile associated with direct transcriptional activation by GR.

Tamoxifen may also upregulate expression of TGF-beta through itsinteraction with the NFkB transcription factor complex, as suggested bythe following observations. (1) The p68 Re1B knockout mouse has aphenotype similar to the TGF-beta knockout mouse, suggesting that Re1Bmay be important in the upregulation of TGF-beta that normally turns offacute inflammation, and (2) the kB-like element in the rat TGF-beta-1promoter is implicated in the tamoxifen-induced stimulation of TGF-betaexpression. Thus, it is likely that a second consequence of ER/NFkBmodulation by these agents is upregulation of TGF-beta expression. It iswell known that TGF-beta has anti-inflammatory and immune-suppressivefunctions. Thus, the induction of TGF-beta by ER/NFkB modulating agentsmay act to synergistically reduce inflammation.

For ER/NFkB modulators, such as idoxifene, several exemplary dosingregimens are contemplated depending upon the particular autoimmunedisease being treated and the stage to which the condition hasprogressed. For the treatment of incipient or early stage rheumatoidarthritis, when inflammation is evident but tissue damage is minimal, alow chronic oral dose of about 0.05 to about 10, preferably about 0.1mg/kg/day, is employed. For local delivery, it is preferred that about0.01 to about 1000 microgram per ml is administered, followed by chroniclow dose oral delivery. When the disease progression is more severe, itis contemplated that a large loading dose, e.g., in the range of about10 to about 100 mg/kg, is used to rapidly establish a therapeutic levelof the ER/NFkB modulator in the circulation, followed by low chronicoral doses.

For the treatment of multiple sclerosis, an exemplary dose regimen is asingle pre-loading dose, e.g., between about 10 to about 100 mg/kg, toestablish a therapeutically effective amount of ER/NFkB modulator in thecirculation, followed by a dose of about 0.1 to about 20, preferablyabout 0.5 to about 5, mg/kg/day.

ER/NFkB modulators that act to reduce or inhibit pathologicalinflammation associated with autoimmune disorders can be identified bythe methods described hereinabove. Specifically, the agents may beidentified by their ability to bind to NFkB/ER complexes, to inhibitNFkB activation induced by TNF-alpha and/or other pro-inflammatorycytokines, and to prevent activation of autoreactive T lymphocytes.

EXAMPLE VIII Effects of the Therapeutic Agents on Cholesterol Levels

Twenty six patients with high cholesterol were administered simvastatinfor 16 weeks. Blood was collected at six times points during the 16weeks and analyzed for TGF-beta levels. While serum cholesterol levelswere reduced in these patients, there was no effect on TGF-beta levelsin any of the patients. In contrast, some of the patients participatingin a trial in which tamoxifen, a tamoxifen analog, or placebo, wasadministered, showed significant decreases in cholesterol levels.Therefore, a combination of one of the therapeutic agents of theinvention and an agent which lowers serum cholesterol levels may exert asynergistic effect and thus, may be useful in the practice in themethods of the invention. Moreover, therapeutic agents of the inventionalone may be useful to lower serum cholesterol levels.

EXAMPLE IX Assay for Measuring Free Anti-sRII Antibody in Human Serum

Recombinant sRII was coated onto the bottom of high protein bindingELISA plates for two hours in 50 mM carbonate buffer (pH 9), thenwashed, and non-specific binding blocked using 5% Tween-20 in 5% sucrosein water, containing 0.02% sodium azide (TSA block). Various serum andplasma samples were then incubated with the coated wells for 2 hours atroom temperature with shaking. Unbound serum components were washed offusing TBS plus 0.05% Tween-20 with four washing cycles ensuring completeaspiration of the well between each cycle. Any bound humanimmunoglobulin was then detected by adding anti-human-IgG antibodiescoupled to horseradish peroxidase in wash buffer for one hour. Boundperoxidase was visualized using TMB substrate. All normal sera testedcontained detectable levels of IgG antibodies binding to sRII. Thissignal was eliminated by omitting any one of: sRII antigen, serum oranti-human-IgG peroxidase. This confirms the presence of high affinityautoantibodies directed against the extracellular domain of the humantype II TGF-β receptor.

It was also determined that human sera from normal healthy individualsdoes not contain autoantibodies against a wide variety of other humanproteins, including, but not limited to, fibrinogen, factor II,compliment C4, apolipoprotein (a), collagen type I, III and IV or theextracellular domain of the human IL-10 receptor (a receptor expressedon endothelial cells). This data suggests that in normal humans there isa relatively specific autoimmune response to the TGFβ type II receptorextracellular domain.

Antibodies of the IgD class against sR11 are also present in normalhuman serum. Although there may be antibodies of the IgM class,interference from rheumatoid factor (IgM directed against IgG) cannot beexcluded at this time. Analysis of the anti-sRII IgG using IgG sub-classspecific detection antisera has demonstrated that the majority of IgGreacting with sRII in normal human serum is of the IgG2 sub-class. Thus,to measure antibodies against sRII, ELISA plate wells are coated withrecombinant or purified human sRII or an immunogenic portion thereof.Wells are blocked against non-specific binding using a blocking agentfor the particular sample type, e.g. for serum analysis, a TCA block.Serum is added to the well, preferably undiluted and untreated. Plasmaor other bodily fluids may also be used. The wells are washed to removeunbound components and the bound anti-sRII Ig is detected using anappropriate anti-human Ig antiserum, labeled for detection.

Because the assay does not determine absolute levels of antibody, thesignal is referenced to a large pool of normal serum (PNS). A standardcurve is constructed for each assay using sequential dilutions of PNS.PNS is arbitrarily designated to have 100 units of anti-sRII Ig, e.g.,IgG.

Results: Healthy normals: Median 100 units 95% of individuals in therange 50 to 200 units NCAs: Median 120 units 95% of individuals in therange 50 to 250 units TVDs: Median 15 units 95% of individuals in therange <10 to 50 units

A result of <50 units on the detection of human pan IgG indicates thepresence of atherosclerosis, with high sensitivity (>95%) and probablysimilar specificity. However, it is envisioned that the detection ofother classes or subclasses, IgG2, may be useful to detect diseasescharacterized by endothelial cell activation, or a specific disease.

Body fluids that contain detectable levels of immunoglobulin may beused, e.g., plasma or serum. Samples can be fresh or frozen. Theanti-sRII Ig are stable over time for a given individual (intrapersonvariation on a 3-month time scale is <10% of the interperson variation).Accurate diagnosis can therefore be achieved on single sample from agiven individual. Moreover, the Ig are stable to multiple cycles offreeze thawing and to long storage times at −20° C. However, the assayis still subject to capture interference by subclass or classes ofimmunoglobulin not otherwise detected. For example, when usinganti-human-IgG perioxidase as the detection antiserum, the assay maydetect little or no IgG against sRII because of the presence of largeamounts of IgD against sRII occupying all the available antigen sites.

The levels of anti-sRII-IgG in serum and plasma samples derived fromindividuals with severe coronary atherosclerosis, defined by coronaryangiography and individuals with normal coronary angiograms, weremeasured. Patients with atherosclerosis (TVD patients) had approximatelya five fold lower median concentration of anti-sRII-IgG compared withindividuals with normal coronary arteries (NCA individuals). Theabsolute amount of sRII IgG could not readily be determined, but therelative amounts compared with pooled normal serum could be determinedby running various dilutions of pooled normal serum as a standard curvewith each assay. In all cases a standard pooled serum was used and thisserum was arbitrarily designated to have 100 units of anti-sRII IgG.

Based on this standardization, the median concentration of anti sR11 IgGamong 100 individuals with coronary atherosclerosis was 14.6 units,compared with 84.9 units among the individuals with normal coronaryarteries. This difference was highly statistically significant (p<0.001;Mann-Whitney U-test). The detection limit for the ELISA as performedunder these conditions was approximately 10 units of anti-sRII-IgG. As aconsequence, fully 40% of the patients with atherosclerosis had levelsat or below the detection limit of the assay, whereas all of theindividuals with normal coronary arteries had detectable levels. Thesensitivity and specificity of this test are estimated to be greaterthan 90%. As a result, measurement of anti-sRII IgG using this assay hasfar greater diagnostic potential than any existing plasma or serumbiochemical marker for coronary heart disease.

This method can conveniently be used to diagnosis the presence of thedisease (e.g. athrosclerosis), determine the extent of disease, evaluateprognosis (i.e, determine future risk prior to onset of symptoms), or tomonitor the effectiveness of a treatment.

The suppressed levels of anti-sRII IgG in plasma and serum fromindividuals with atherosclerosis may be due to (a) lower levels ofanti-sRII IgG, which assumes that lower detection of anti-sRII IgGresults from the presence of lower levels of the IgG; (b) increasedlevels of anti-sRII IgD, or other non IgG classes, as the assays aresubject to inhibition by non-IgG class anti-sRII antibodies; or (c)increased levels of sRII antigen. The sRII antigen which is normallyexpressed on endothelial cells may be shed during phenotypic changes inendothelial cell gene expression pattern, e.g., during activation, aprocess thought to occur in atherogenesis. sRII in plasma would thenform complexes with the anti-sRII antibodies and make them moredifficult to detect. As a result, lower levels of anti-sRII IgG would bedetectable in individuals with increased endothelial cell activation.

MacCaffrey and colleagues have reported a switch from expression ofTGF-β type II receptor to TGF-β type I receptor during the developmentof atherosclerosis in man, and one mechanism which might contribute tothis switch would be shedding of sRII from endothelial cells as theybecome activated (MacCaffrey et al. J. Clin. Invest. 1996, 2667-2675).Thus, plasma concentrations of sRII may be a direct measure of the stateof endothelial cell activation (related, for example, to functionaltests of endothelial cells function, e.g., brachial reactivity). Sincethe presence of sRII is expected to reduce detection of anyanti-sRII-IgG present in the plasma (by forming complexes with it), thelevel of anti-sRII-IgG would be a proxy measure for levels sRII antigen(i.e., low levels of anti-sRII-IgG result from high levels of sRIIantigen, resulting, in turn, from endothelial cell activation). Thus,this assay represents the first useful plasma measure of endothelialcell function, and thus, is a measure of an individual at risk of orhaving a disease characterized by endothelial cell activation. Moreover,the assay offers many advantages over the low throughput endothelialcell function assays such as brachial reactivity currently being used.

In addition to the assay described above, the metholodology describedherein can also be utilized to carry out the following assays:

(a) Detection of free sRII antigen High levels diagnostic for verysevere atherosclerosis (b) Detection of sRII: anti-sRII High leveldiagnostic of moderate to complexes very severe atherosclerosis (c)Detection of total sRII antigen Diagnostic of extent of endothelial cellactivation and hence of athero- sclerotic disease progression.

The methodology described herein can also be used to determine the level(e.g. the relative presence or absence) of TGF-β type II receptors (e.g.the extracellular domain of the TGF-β type II receptor) in mammaliancells or tissue. Endothelial cells are believed to shed theextracellular domain of the TGF-β type II receptor during activation,and there is believed to be a correlation between endothelial cellactivation and atherogenesis, as well as other diseases. Accordingly,the invention also provides a method comprising detecting TGF-β type IIreceptors in mammalian cells or tissue, by combining the cells or tissuewith a capture moiety that binds TGF-β type II receptors or a portionthereof, forming a capture complex, and detecting or determining theamount of the capture complex.

All publications and patents are incorporated by reference herein, asthough individually incorporated by reference, as long as they are notinconsistent with the present disclosure. The invention is not limitedto the exact details shown and described, for it should be understoodthat many variations and modifications may be made while remainingwithin the spirit and scope of the invention defined by the claims.

What is claimed is:
 1. A method of treating-a mammal having, or at riskof, an indication associated with a TGF-beta deficiency comprisingadministering an amount of an aspirinate effective to elevate the levelof TGF-beta; wherein the indication is not cancer, arthritis,hypertension, angina, multiple sclerosis, or lupus erythermatosis.
 2. Amethod of treating a mammal having, or at risk of, a vascular indicationwhich is associated with a TGF-beta deficiency, comprising administeringan amount of an aspirinate effective to elevate the level of TGF-beta soas to inhibit or reduce the diminution of vessel lumen diameter; whereinthe vascular indication is not hypertension or angina.
 3. A method oftreating a mammal having, or at risk of, an indication associated with aTGF-beta deficiency other than hypertension, thrombosis oratherosclerosis, comprising administering an amount of3-acetoxy-2-carboxythiophene or a pharmaceutically acceptable saltthereof, effective to elevate the level of TGF-beta.
 4. The method ofclaim 2 wherein the administration is effective to reduce or preventlipid accumulation by the vessel, to increase plaque stability of anatherosclerotic lesion, to inhibit atherosclerotic lesion formation ordevelopment, or to induce atherosclerotic lesion regression.
 5. Themethod of claim 1 or 2 wherein the aspirinate is not3-acetoxy-2-carboxythiophene or a pharmaceutically acceptable saltthereof.
 6. A therapeutic method for preventing or treating a conditionor symptom associated with Parkinson's disease, Marfan's syndrome,Alzheimer's disease, senile dementia, osteoporosis, or fibrosis,comprising administering to a mammal in need of such therapy, aneffective amount of an aspirinate.
 7. A therapeutic method forpreventing or treating a condition or symptom associated with anauto-immune disease, comprising administering to a mammal in need ofsuch therapy, an effective amount of an aspirinate, provided theasprinate is not a copper salt of an aryl or heteroaryl carboxylic acid.8. A therapeutic method for lowering serum cholesterol, comprisingadministering to a mammal in need of such therapy, an effective amountof an aspirinate.
 9. A therapeutic method for enhancing or promotingwound healing, comprising administering to a mammal in need of suchtherapy, an effective amount of an aspirinate.
 10. A method of treatinga mammal having, or at risk of, an indication associated with a TGF-betadeficiency comprising administering an amount of an aspirinate effectiveto elevate the level of TGF-beta; wherein the aspirinate is a compoundof formula (I):

wherein R¹ is hydrogen, halo, nitro, cyano, hydroxy, CF₃, —NR_(c)R_(d),—C(═O)OR_(e), —C(═N)OR_(e) —OC(═O)OR_(e), (C₁-C₆)alkyl or (C₁-C₆)alkoxy;R² is hydrogen or —XR_(a); R³ is —C(═O)YR_(b), or —N(R_(f))C(═O)R_(g)—;R⁴ is (═O)_(n); or R⁴ is (C₁-C₆)alkyl, (C₁-C₆)alkanoyl or(C₂-C₆)alkanoyloxy; R⁵ is hydrogen, —C(═O)OR_(h) or —C(═O)SR_(h); n is0, 1 or 2; X is oxygen, —N(R_(i))—, or sulfur; Y is oxygen or sulfur;R_(a) is (C₁-C₆)alkanoyl, (C₁-C₆)alkyl, or hydrogen; R_(b) is hydrogenor (C₁-C₃)alkyl; R_(c) and R_(d) are each independently hydrogen,(C₁-C₄)alkyl, phenyl, C(═O)OH, C(═O)O(C₁-C₄)alkyl CH₂C(═O)OH,CH₂C(═O)O(C₁-C₄)alkyl, or (C₁-C₄)alkoxy; or R_(c) and R_(d) togetherwith the nitrogen to which they are attached are a 3, 4, 5, or 6membered heterocyclic ring; and R_(e)-R_(i) are independently hydrogenor (C₁-C₆)alkyl; or a pharmaceutically acceptable salt thereof providedthat R² and R³ are on adjacent positions of the ring to which they areattached, or are on the 2- and 5-positions of the ring; and furtherprovided that when R² is hydrogen; R³ is on the 2-or 5-position of thering to which it is attached and R⁴ is (C₁-C₄)alkanoyloxy.
 11. A methodof treating a mammal having, or at risk of, an indication associatedwith a TGF-beta deficiency comprising administering an amount of anaspirinate effective to elevate the level of TGF-beta; wherein theaspirinate is 3,5-diisopropyl salicylic acid, salicylic acid,3,5-di(tertiarybutyl)salicylic acid, adamantylsalicylic acid,3,5-dibromoacetylsalicylic acid, 3,5-diiodoacetylsalicylic acid,4-(tertiarybutyl)salicylic acid, 4-nitrosalicylic acid, 4-aminosalicylicacid, 4-acetylaminosalicylic acid, 5-chlorosalicylic acid, or3,5-dichlorosalicylic acid, or a pharmaceutically acceptable saltthereof, provided that the salt is not a copper salt.
 12. A method oftreating a mammal having, or at risk of, an indication associated with aTGF-beta deficiency comprising administering an amount of an aspirinateeffective to elevate the level of TGF-beta; wherein the aspirinate isnot a copper salt; and wherein the indication is not hypertension orangina.
 13. A method of treating a mammal having, or at risk of, avascular indication which is associated with a TGF-beta deficiency,comprising administering an amount of an aspirinate effective to elevatethe level of TGF-beta so as to inhibit or reduce the diminution ofvessel lumen diameter; wherein the aspirinate is not a copper salt; andwherein the vascular indication is not hypertension or angina.
 14. Themethod of claim 13 wherein the administration is effective to reduce orprevent lipid accumulation by the vessel, to increase plaque stabilityof an atherosclerotic lesion, to inhibit atherosclerotic lesionformation or development, or to induce atherosclerotic lesionregression.
 15. The method of claim 12 or 13 wherein the aspirinate isnot 3-acetoxy-2-carboxythiophene or a pharmaceutically acceptable saltthereof.
 16. A therapeutic method for preventing or treating a conditionor symptom associated with Parkinson's disease, Marfan's syndrome,Alzheimer's disease, senile dementia, osteoporosis, multiple sclerosis,lupus erythermatosis, or fibrosis, comprising administering to a mammalin need of such therapy, an effective amount of an aspirinate; whereinthe aspirintate is not a copper salt.
 17. The method of claim 8 whereinthe aspirinate is not a copper salt.
 18. The method of claim 9 whereinthe aspirinate is not a copper salt.